tacrolimus and Reperfusion-Injury

Liver transplantation has been widely accepted as an effective intervention for end-stage liver diseases and early hepatocellular carcinomas. However, a variety of postoperative complications and adverse reactions have baffled medical staff and patients. Currently, transplantation monitoring relies primarily on nonspecific biochemical tests, whereas diagnosis of multiple complications depends on invasive pathological examination. Therefore, a noninvasive monitoring method with high selectivity and specificity is desperately needed. This review summarized the potential of endogenous small-molecule metabolites as biomarkers for assessing graft function, ischemia-reperfusion injury and liver rejection. Exogenous metabolites, mainly those immunosuppressive agents with high intra- and inter-individual variability, were also discussed for transplantation monitoring.

Topics: Biomarkers; Carcinoma, Hepatocellular; Everolimus; Graft Rejection; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Metabolome; Mycophenolic Acid; Postoperative Complications; Prognosis; Reperfusion Injury; Tacrolimus

Tacrolimus was used as a rinse solution against ischaemia-reperfusion injury (IRI) in liver transplantation for years but its protective effects remain controversies.. We conducted literature retrieval in electronic databases including MEDLINE, EMBASE and Cochrane Central to identify relevant randomized controlled trials (RCTs) investigating the effects of tacrolimus as a rinse solution in liver transplantation. Postoperative liver function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL), at postoperative day (POD) 1, 2 and 7 was extracted for pooled estimation. Forest plots were generated to calculate the differences between the groups. The I2 index statistic was used to assess heterogeneity. Publication bias was evaluated using funnel plots and Egger's test.. Three RCTs including 70 liver transplants were evaluated in this study. Pooled estimation revealed that rinse with tacrolimus in liver transplantation did not provide hepatic protection with respect to postoperative ALT (Test Z = 1.36; P = .175), AST (Test Z = 1.70; P = .090) or TBIL (Test Z = 0.69; P = .490). Sensitivity analysis by excluding extended donor criteria (EDC) livers showed similar results. Funnel plots and Egger's test demonstrated that there was no substantial bias.. We may tentatively conclude that tacrolimus is ineffective for amelioration of postoperative liver function as a rinse solution in liver transplantation. Nevertheless, there is great space for future research in this area, and the potential clinical value of tacrolimus needs to be further addressed. We are expecting more evidence to support our speculations.

Topics: Alanine Transaminase; Aspartate Aminotransferases; Bilirubin; Humans; Liver; Liver Transplantation; Pharmaceutical Solutions; Postoperative Period; Protective Agents; Randomized Controlled Trials as Topic; Reperfusion Injury; Tacrolimus; Treatment Outcome

Endoplasmic reticulum (ER) stress is a situation caused by the accumulation of unfolded proteins in the endoplasmic reticulum, triggering an evolutionary conserved adaptive response termed the unfolded protein response. When adaptation fails, excessive and prolonged ER stress triggers cell suicide. Important roles for ER-initiated cell death pathways have been recognized for several diseases, including diabetes, hypoxia, ischemia/reperfusion injury, neurodegenerative and heart diseases. The implication of the ER stress is not well recognized in solid organ transplantation, but increasing evidence suggests its implication in mediating allograft injury. The purpose of this review is to summarize the mechanisms of ER stress and to discuss its implication during tissue injury in solid organ transplantation. The possible implications of the ER stress in the modifications of cell functional properties and phenotypic changes are also discussed beyond the scope of adaptation and cell death. Increasing the understanding of the cellular and molecular mechanisms of acute and chronic allograft damages could lead to the development of new biomarkers and to the discovery of new therapeutic strategies to prevent the initiation of graft dysfunction or to promote the tissue regeneration after injury.

Topics: Animals; Apoptosis; Cyclosporine; Diabetes Mellitus, Type 2; Drug Delivery Systems; Endoplasmic Reticulum; Graft Survival; Humans; Insulin Resistance; Islets of Langerhans; Kidney Transplantation; Liver Transplantation; Mice; Organ Preservation; Postoperative Complications; Reperfusion Injury; Tacrolimus; Transplantation; Transplantation, Homologous; Unfolded Protein Response

Oxidative stress causes disturbances in homeostasis leading to an excessive production of reactive oxygen species (ROS). Free-radical reactions undergo intensification during transplantation and are responsible for both damage to the graft and cardiovascular complications, one of the major causes of patient death. The function of immunosuppressive drugs in this process is currently the object of research. Investigations of medications which would decrease the level of oxidative stress are in progress. Tacrolimus (FK-506) is a medication commonly used in immunosuppressive therapy. It has a better cardiac-lipid profile than cyclosporine A. Some reports about the beneficial effect of tacrolimus on the level of oxidative stress in the organism have appeared. Especially in vitro studies and animal tests indicate antioxidative properties for tacrolimus. Decreases in parameters of oxidative stress, such as the concentration of malone dialdehyde (MDA), the activity of myeloperoxidase (MPO), and neutrophilic infiltration, were observed after treatment. In in vitro studies on endotheliocytes, tacrolimus induced oxidative stress more weakly than other medications and was the only one that did not increase the production of nitric oxide (NO). The protective effect of tacrolimus on inflammatory response in rat liver during ischemia-reperfusion injury, on atrocytes exposed to stimulated ischemia in vitro, and in experimental traumatic injury of spinal cord tissues in rats were also described. Findings in patients after transplantation are not so clear and even indicate that the influence of tacrolimus on the activity of antioxidative enzymes in kidneys may be involved in side-effect of tacrolimus.

Topics: Animals; Disease Models, Animal; Free Radical Scavengers; Humans; Immunosuppressive Agents; Kidney; Liver; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Tacrolimus

In addition to efficacious immunosuppression for the benefit of organ transplantation, tacrolimus has diverse actions that result in amelioration of ischemia-reperfusion injury. Knowledge is accumulating rapidly on the mechanisms through which tacrolimus exerts these cytoprotective effects, including alterations in microcirculation, free radical metabolism, calcium-activated pathways, inflammatory cascades, mitochondrial stability, apoptosis, stress-response proteins, and tissue recovery. Within the nucleus, actions mediating the effects of tacrolimus appear to be dominantly influenced by interactions with the transcription factor, nuclear factor-kappaB. Because tacrolimus is a cornerstone agent in immunosuppression regimens throughout the world and knowledge of its cellular mechanisms is evolving, it is important to update the clinical literature with this information. We reviewed the published literature with intent to portray the interactions of tacrolimus in the intricate cellular mechanisms initiated by ischemia and reperfusion.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Calcium; Cell Communication; Cytokines; Free Radicals; Humans; Immunosuppressive Agents; Microcirculation; Neutrophils; Platelet Activation; Reperfusion Injury; Tacrolimus

NF-kappaB is an inducible nuclear transcription factor regulating the expression of many genes. NF-kappaB activation may function as a master switch in a variety of immune and inflammatory processes, including sepsis and transplant tolerance. In this review, we summarize features of NF-kappaB regulation, as well as describe its role in intracellular signal transduction pathways. Subsequently, we concentrate on the role of NF-kappaB in the field of organ transplantation and the role of NF-kappaB in organ ischemia/reperfusion injury and graft rejection. Finally, potential therapeutic strategies are discussed to modify NF-kappaB activity with certain immunosuppression medications, including cyclosporine, tacrolimus, and glucocorticoids.

Topics: Adenoviridae; Animals; Cyclosporine; Gene Expression Regulation; Genetic Therapy; Graft Rejection; Hepatitis C, Chronic; Humans; Immunosuppressive Agents; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oligodeoxyribonucleotides; Organ Transplantation; Reperfusion Injury; Signal Transduction; Sulfasalazine; Tacrolimus

Brain ischemia produces morphologic and biochemical alterations in astrocytes. This mini-review summarizes astrocytic responses to brain ischemia including our studies on the neuronal and astrocytic Na(+)-Ca2+ exchanger (NCX). NCX is considered to cause Ca2+ efflux (forward mode) or Ca2+ influx (reverse mode), depending on the electrochemical gradient of Na+ across the plasma membranes and membrane potential. We demonstrated that NCX is present in cultured neurons and astrocytes and that there are differences in their properties and distribution ratio of the isoforms between neurons and astrocytes. We also found that Ca2+ depletion followed by reperfusion with Ca(2+)-containing medium caused cell death in cultured astrocytes (Ca2+ paradox-like injury), but not in neurons. The study, carried out by the use of a specific antisense oligomer, provides direct evidence that Ca2+ paradox-like injury is mediated by NCX in the reverse mode. The injury was attenuated by inhibitors of the Na(+)-Ca2+ exchanger, heat shock protein and the calcineurin inhibitor FK506. In a preliminary experiment, we found that brain ischemia decreases the mRNA level of NCX in the hippocampus. Further studies on activation and cell injury of astrocytes will contribute to development of new drugs that modulate the function of astrocytes.

Topics: Animals; Astrocytes; Brain; Brain Ischemia; Calcineurin; Calcineurin Inhibitors; Calcium; Cell Death; Cells, Cultured; Heat-Shock Proteins; Humans; Immunosuppressive Agents; Membrane Potentials; Neurons; Reperfusion Injury; Sodium-Calcium Exchanger; Tacrolimus

The increased use of older and/or marginal donor organs in liver transplantation over the last decade calls for strategies to minimize ischaemic reperfusion (I/R) injury to prevent early graft failure. Tacrolimus, a very potent and effective calcineurin inhibitor, was selected because of its ability to ameliorate I/R injury. A randomized, blinded, controlled single-centre trial of 26 liver transplant recipients was performed between February 2008 and December 2009. Donor organs were randomized to be perfused intraportally during liver transplantation with 1.5 l 5% albumin infusion containing either 20 ng/ml tacrolimus or placebo. The primary end point was liver function as assessed by aspartate transaminase (AST) or alanine transaminase (ALT) levels 6 days after transplantation. Treatment effectiveness was tested by transcriptome-wide analysis of biopsies. There was no difference in the primary end point, i.e. AST (IU/l) and ALT (IU/l) at day 6 after transplantation between groups. Furthermore, choleastatic parameters as well as parameters of liver synthesis were not different between groups. However, tacrolimus treatment suppressed inflammation and immune response in the transplanted liver on a genome-wide basis. Intrahepatic administration of tacrolimus did not result in a reduction of AST and ALT within the first week after transplantation.

Topics: Adult; Alanine Transaminase; Aspartate Aminotransferases; Female; Gene Expression Profiling; Graft Rejection; Hepatitis; Humans; Infusions, Intravenous; Liver; Liver Transplantation; Male; Middle Aged; Portal Vein; Reperfusion Injury; Tacrolimus

Interventions that minimize hepatic ischemia/reperfusion injury (IRI) can expand the donor organ pool. Thymoglobulin (TG) induction therapy has been shown to ameliorate delayed graft function and possibly decrease IRI in cadaver renal transplants recipients. This controlled randomized trial was designated to assess the ability of TG to protect against IRI in liver transplant recipients.. Twenty-two cadaveric liver transplant recipients were randomized to receive either TG (1.5 mg/kg/dose) during the anhepatic period and QOD x2 doses or no TG. No differences in recipients' demographics were present and donor characteristics were similar in terms of age, cause of death, and cold ischemia time. Maintenance immunosupression consisted of Tacrolimus (or Cyclosporine) and steroids for both groups. Donor biopsies were obtained during organ procurement, cold storage and 1 h after re-vascularization. Post-operative liver function tests were monitored. Early graft function, length of stay, patient and graft survival rates, incidence of primary non-function and rate of rejection were assessed.. Patient and graft survival at 3 months was 100%. There was no incidence of primary graft non-function and no need for re-transplantation. The incidence of acute rejection was similar between the two groups. Patients in the TG group had significant decreases in alanine aminotransferase test at day 1 compared to the control group (p = 0.02). There were also near significant decreases of total bilirubin at day 5 and shorter length of hospitalization. Liver biopsy (at procurement, when cold, and post-reperfusion) of TG group demonstrated a trend for increased central ballooning.. The TG allowed for more compromised liver grafts to be transplanted with less clinical evidence of IRI and improved function. Further studies on the degree of apoptosis in the liver biopsy post-reperfusion are underway.

Topics: Adult; Aged; Antilymphocyte Serum; Bilirubin; Female; Graft Rejection; Humans; Immunosuppressive Agents; Length of Stay; Liver; Liver Function Tests; Liver Transplantation; Male; Middle Aged; Reperfusion Injury; Survival Rate; Tacrolimus; Transaminases; Transplantation, Homologous

The goal of this report is to evaluate in a prospective randomized fashion the effect of flushing hepatic allografts with tacrolimus before transplantation. A prospective, double-blinded, randomized trial was performed. Twenty patients receiving orthotopic liver transplants from October 2000 to October 2001 were randomized into two groups. Group 1 (active) was administered tacrolimus, 20 ng/mL, plus Plasma-lyte A (Baxter Healthcare Corp, Deerfield, IL) liver flush solution; and group 2 (placebo) was administered only Plasma-lyte A. Ischemia/reperfusion injury was assessed in both groups after transplantation by means of serum laboratory values to assess hepatocellular damage, synthetic function, and ion transport capacity. Peak values were recorded for each parameter, and their distributions were compared. There were no statistically significant differences between groups for age, sex, total ischemia time, or cause of liver disease. Global multivariate comparison of peak changes in all measures of liver function indicated liver injury was significantly lower with tacrolimus treatment than placebo (P =.01). The sample median for group 1 was less than for group 2 in all parameters measured. Individual statistical comparison showed that peak changes from baseline aspartate aminotransferase and activated partial thromboplastin time values were significantly improved (P

Topics: Adolescent; Adult; Aged; Bilirubin; Double-Blind Method; Female; Humans; Immunosuppressive Agents; Liver Transplantation; Male; Middle Aged; Prothrombin Time; Reperfusion Injury; Solutions; Tacrolimus

This study tested the hypothesis that inflammatory and interleukin (IL)-17 signalings were essential for acute liver ischemia (1 h)-reperfusion (72 h) injury (IRI) that was effectively ameliorated by adipose-derived mesenchymal stem cells (ADMSCs) and tacrolimus.. Adult-male SD rats (n = 50) were equally categorized into groups 1 (sham-operated-control), 2 (IRI), 3 [IRI + IL-17-monoclonic antibody (Ab)], 4 (IRI + tacrolimus), 5 (IRI + ADMSCs) and 6 (IRI + tacrolimus-ADMSCs) and liver was harvested at 72 h.. The main findings included: (1) circulatory levels: inflammatory cells, immune cells, and proinflammatory cytokines as well as liver-damage enzyme at the time point of 72 h were highest in group 2, lowest in group 1 and significantly lower in group 6 than in groups 3 to 5 (all p < 0.0001), but they did not differ among these three latter groups; (2) histopathology: the liver injury score, fibrosis, inflammatory and immune cell infiltration in liver immunity displayed an identical pattern of inflammatory cells among the groups (all p < 0.0001); and (3) protein levels: upstream and downstream inflammatory signalings, oxidative-stress, apoptotic and mitochondrial-damaged biomarkers exhibited an identical pattern of inflammatory cells among the groups (all p < 0.0001).. Our results obtained from circulatory, pathology and molecular-cellular levels delineated that acute IRI was an intricate syndrome that elicited complex upstream and downstream inflammatory and immune signalings to damage liver parenchyma that greatly suppressed by combined tacrolimus and ADMSCs therapy.

Topics: Animals; Interleukin-17; Liver Diseases; Male; Mesenchymal Stem Cells; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Delivery of cerebroprotective agents using liposomes has been demonstrated to be useful for treating cerebral ischemia/reperfusion (I/R) injury. We previously reported that intravenous administration of liposomes with diameters of 100 nm showed higher accumulation in the I/R region compared with larger liposomes (>200 nm) by passage through the disintegrated blood-brain barrier, suggesting a size-dependence for liposome-mediated drug delivery. Based on these findings, we hypothesized that regulation of liposomal particle size (<100 nm) may enhance the therapeutic efficacy of encapsulated drugs on cerebral I/R injury. Herein, we prepared lipid nanoparticles (LNP) with particle sizes <100 nm by the microfluidics method and compared their therapeutic potential with LNP exhibiting sizes >100 nm in cerebral I/R model rats. Intravenously administered smaller LNP (ca. 60 nm) exhibited wider accumulation and diffusivity in the brain parenchyma of the I/R region compared with larger LNP (>100 nm). Importantly, treatment with LNP encapsulating the cerebroprotective agent FK506 (FK-LNP) with particle sizes <100 nm showed greater cerebroprotective effects than FK-LNP with sizes >100 nm, and also significantly ameliorated brain injury. These results suggest that particle size regulation of LNP to sizes <100 nm can enhance the therapeutic effect of encapsulated drugs for treatment of cerebral I/R injury, and that FK-LNP could be a promising cerebroprotective agent.

Topics: Animals; Brain Ischemia; Liposomes; Nanoparticles; Neuroprotective Agents; Particle Size; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus

Topics: Animals; Brain Death; Calcineurin Inhibitors; Lung; Lung Injury; Reperfusion Injury; Swine; Tacrolimus

Acute kidney injury (AKI) is a major cause of patient mortality and a major risk multiplier for the progression to chronic kidney disease (CKD). The mechanism of the AKI to CKD transition is complex but is likely mediated by the extent and length of the inflammatory response following the initial injury. Lymphatic vessels help to maintain tissue homeostasis through fluid, macromolecule, and immune modulation. Increased lymphatic growth, or lymphangiogenesis, often occurs during inflammation and plays a role in acute and chronic disease processes. What roles renal lymphatics and lymphangiogenesis play in AKI recovery and CKD progression remains largely unknown. To determine if the increased lymphatic density is protective in the response to kidney injury, we utilized a transgenic mouse model with inducible, kidney-specific overexpression of the lymphangiogenic protein vascular endothelial growth factor-D to expand renal lymphatics. "KidVD" mouse kidneys were injured using inducible podocyte apoptosis and proteinuria (POD-ATTAC) or bilateral ischemia reperfusion. In the acute injury phase of both models, KidVD mice demonstrated a similar loss of function measured by serum creatinine and glomerular filtration rate compared to their littermates. While the initial inflammatory response was similar, KidVD mice demonstrated a shift toward more CD4+ and fewer CD8+ T cells in the kidney. Reduced collagen deposition and improved functional recovery over time was also identified in KidVD mice. In KidVD-POD-ATTAC mice, an increased number of podocytes were counted at 28 days post-injury. These data demonstrate that increased lymphatic density prior to injury alters the injury recovery response and affords protection from CKD progression.

Topics: Acute Kidney Injury; Animals; Apoptosis; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Collagen; Disease Models, Animal; Kidney; Lymphangiogenesis; Lymphatic Vessels; Mice; Mice, Transgenic; Podocytes; Proteinuria; Recovery of Function; Reperfusion Injury; Tacrolimus; Vascular Endothelial Growth Factor D

Tacrolimus and mycophenolate mofetil are immunosuppressive agents widely used on the postoperative period of the transplants.. To evaluate the influence of the association of them on the abdominal wall healing in rats.. Thirty-six Wistar rats were randomly assigned in three groups of 12. On the early postoperative period, four of the control group and three of the experimental groups died. The three groups were nominated as follow: control group (GC, n=8); group I (GI, n=11, standard operation, mycophenolate mofetil and tacrolimus); group II (GII, n=10, standard operation, mycophenolate mofetil and tacrolimus). The standard operation consisted of right total nephrectomy and 20 min ischemia of the left kidney followed by reperfusion. Both NaCl 0.9% and the immunosuppressive agents were administered starting on the first postoperative day and continuing daily until the day of death on the 14th day. On the day of their deaths, two strips of the anterior abdominal wall were collected and submitted to breaking strength measurement and histological examination.. There were no significant differences in wound infection rates (p=0,175), in the breaking strength measurement and in the histological examination among the three groups.. The combination of the immunosuppressive agents used in the study associated with renal ischemia and reperfusion does not interfere in the abdominal wall healing of rats.

Topics: Abdominal Wall; Animals; Immunosuppressive Agents; Ischemia; Kidney; Mycophenolic Acid; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Tacrolimus

Acute kidney injury (AKI) is commonly encountered and causes high mortality in hospitalized patients; however, effective therapies for AKI have still not been established. Accordingly, we performed a rodent model with acute renal ischemia-reperfusion (IR) and tested the hypothesis that combined tacrolimus and melatonin therapy could be superior to either one for protecting the kidney against IR injury. Adult-male SD rat (n = 30) were equally categorized into group 1 (receiving laparotomy only), group 2 (IR treated by 3.0 cc/normal-saline), group 3 [IR + tacrolimus/0.5 mg/kg by intravenous administration at 30 minutes and at days 1/2/3 after IR], group 4 (IR + melatonin/50 mg/kg by intra-peritoneal administration at 30 minutes and 25 mg/kg at days 1/2/3 after IR] and group 5 (IR + tacrolimus +melatonin). By day 3 after IR, the creatinine/BUN levels and ratio of urine protein to urine creatinine were highest in group 2, lowest in group 1 and significantly lower in group 5 than in groups 3/4 (all P < .0001), but they did not differ between the groups 3/4. The protein expressions of oxidative-stress (p47phox/NOX-1/NOX-2/NOX-4), upstream (TLR4/MAL/MyD88/TRAF6/ASK1/MKK4/MKK7/NF-κB) and downstream (IL-6/INF-γ/MMP-9/IL-1ß) inflammatory signaling, MAPK-family-signaling cascades(ERK1/2, JNK/p38/c-JUN), apoptotic/autophagic (p53/caspase 3/mitochondrial-Bax, ratio of LC3B-II/LC3B-I), and mitochondrial-damaged (cyclophilin D/cytochrome C/DRP1) biomarkers, and the expressions of inflammatory-immune cells (F4/80, CD14/CD3/CD8) as well as the kidney injured score exhibited an identical pattern of creatinine level (all P < .0001). In conclusion, combined tacrolimus and melatonin therapy was better than either single one on protecting the kidney functional and anatomical integrity against IR injury through suppressing inflammation and the generation of oxidative stress.

Topics: Acute Kidney Injury; Animals; Antioxidants; Drug Therapy, Combination; Immunosuppressive Agents; Male; Melatonin; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.

Topics: Acute Kidney Injury; Animals; Cell Line; Drug Discovery; Human Umbilical Vein Endothelial Cells; Humans; Macrolides; Mice; Protective Agents; Proteome; Reperfusion Injury; Sirolimus; Swine; Tacrolimus; Tacrolimus Binding Proteins; TOR Serine-Threonine Kinases

Hepatic ischemia reperfusion (HIR) has been found to induce brain injury and cognitive dysfunction. Dynamin-related protein 1 (Drp1) mediated mitochondrial fission involves oxidative stress, apoptosis and several neurological diseases. In this study, we investigated whether Drp1 translocation to mitochondria was implicated in HIR induced hippocampus injury in young mice, and further detected the role of calcineurin in the regulation of mitochondrial dynamics. 2-week C57BL/6 mice were chosen to make HIR model. Western blot was used to detect mitochondrial dynamics regulating proteins in whole hippocampal tissues and extracted mitochondria. Transmission electron microscopy was used to observe mitochondrial morphology. TUNEL staining and ELISA (serum S100β/NSE concentrations) were used to evaluate neurons apoptosis and brain injury respectively. Drp1 inhibitor Mdivi-1 and calcineurin inhibitor FK506 were utilized to further confirm the role of Drp1 and calcineurin. Results showed that HIR affected mitochondrial dynamics in a fission-dominant manner with translocation of Drp1 to mitochondria in hippocampus of young mice. HIR induced increased expression of calcineurin and dephosphorylation of Drp1 at Ser637 in hippocampus. Treatment with Mdivi-1 and FK506 upregulated the phosphorylation of Drp1, inhibited Drp1 translocation to mitochondria, and alleviated mitochondrial fragmentation after HIR. What's more, Mdivi-1 and FK506 restrained cytochrome c release and cleaved caspase-3 expression, ameliorated hippocampal neurons apoptosis, and decreased serum S100β/NSE concentrations as well. These data suggest that calcineurin mediated Drp1 dephosphorylation and translocation to mitochondria play a crucial role in HIR induced mitochondrial fragmentation and neurons apoptosis in hippocampus.

Topics: Animals; Apoptosis; Calcineurin; Cytochromes c; Dynamins; Hippocampus; Liver; Mice; Mice, Inbred C57BL; Mitochondria, Liver; Mitochondrial Dynamics; Oxidative Stress; Phosphorylation; Quinazolinones; Reperfusion; Reperfusion Injury; Tacrolimus

The study is aimed to investigate the protective effects and possible mechanism of tacrolimus (FK506) pre-treatment in hepatic ischemia-reperfusion injury in rats.. The rats were randomly assigned into four groups, which were S, IR, L and H group, and then all groups were subjected to 60min of 70% partial warm liver ischemia, except S group. Rats in the L and H group were pre-treated with two different doses FK506 at 60min before ischemia. The rats of the IR group received an identical volume of normal saline. All animals were sacrificed after 6h of reperfusion. Transaminases were measured by biochemistry analyzer. Elisa kit was used to detect TNF-α, IL-6 and IL-1β levels in serum. Liver specimens were stained with hematoxylin and eosin (HE) to assess the pathologic changes. The expressions of heme oxygenase-1 (HO-1), hypoxia-inducible factor-1α (HIF-1α), nuclear factor of activated T cells (NFAT3) were measured by real-time quantitative PCR and western blotting and the Bcl-2 and the Bax protein were tested by western blotting.. In rats pre-treated with FK506, the levels of transaminases, TNF-α and IL-1β were reduced significantly and also liver damage was dramatically mitigated compared to those without FK506 pre-treatment. Moreover, the expression of HO-1 at the level of both transcription and translation increased clearly and the activation of the HIF-1α was found in FK506 pre-treated livers. Moreover, NFAT3 protein transportation to the nucleus was reduced and Bax protein expression was decreased, but the expression of Bcl-2 protein was markedly increased after FK506 pre-treatment.. FK506 pre-treatment could lessen hepatic ischemia-reperfusion injury through up-regulating the expression of HIF-1α and HO-1, and inhibiting nuclear translocation of NFAT3 in liver tissues.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; bcl-2-Associated X Protein; Heme Oxygenase-1; Hypoxia-Inducible Factor 1, alpha Subunit; Immunosuppressive Agents; Interleukin-1beta; Interleukin-6; Liver; Male; NFATC Transcription Factors; Preoperative Care; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus; Time Factors; Tumor Necrosis Factor-alpha

Small-for-size grafts have become more important, especially in living donor liver transplants. The Pringle maneuver, used to reduce blood loss, and the immunosuppressive medications used to prevent graft rejection in liver transplants have different side effects on liver regeneration. We researched the effect of situations where tacrolimus and the Pringle maneuver were applied or not on liver regeneration in rats with partial hepatectomy.. This study was completed with 35 Wistar Albino rats. The subjects were randomly divided into 5 groups: Group 1 had the abdomen opened and no other procedure was performed; Group 2 underwent a 70% hepatectomy; Group 3 underwent a 15-minute Pringle maneuver + 70% hepatectomy; Group 4 underwent a 70% hepatectomy + 5 days of 1 mg/kg/day intraperitoneal tacrolimus; and Group 5 underwent a 150 minute Pringle maneuver + 0% hepatectomy + 5 days of 1 mg/kg/day intraperitoneal tacrolimus. All rats were sacrificed on the seventh postoperative day, remaining liver tissue was weighed, and weight indices created. The remaining liver tissue was stained with phosphohistone H3 and the mitotic index calculated.. The groups that underwent the Pringle maneuver, 70% hepatectomy, and tacrolimus administration were compared with the control group in terms of mitotic index and weight index, but no statistically significant differences were identified.. Suppression of regeneration forms a risk after liver transplantation with small-volume grafts. As a result, research on the effect of tacrolimus combined with the Pringle maneuver is important, especially for transplantations using segmented liver grafts. In our study, we showed that the use of tacrolimus had no negative effect on liver regeneration.

Topics: Animals; Disease Models, Animal; Hepatectomy; Immunosuppressive Agents; Liver Regeneration; Liver Transplantation; Male; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus

Ischemia-reperfusion injury after liver transplantation (LT) impairs graft function and affects prognosis of recipients. Isoglycyrrhizinate magnesium (Iso) is a hepatoprotective drug usually used after liver injury. In this study, we intended to explore whether Iso alone have protective effect after ischemia-reperfusion injury in a rat model of liver transplantation. We also aimed to study whether Iso could enhance the hepatoprotective effect of FK506 (tacrolimus) and underlying mechanism.. Rats after LT were treated with different concentration of FK506 with or without, Iso or lower-dose FK506 plus Iso. Alanine transaminase, aspartate transaminase, and albumin level were measured after 48 hours, 72 hours, and 7 days. A cell ischemic/reperfusion model was established to further study the mechanism of hepatoprotective effect of FK506 and Iso.. Iso treatment alone had no effect on liver grafts after LT, but lower-dose FK506 + Iso was better for maintenance of liver function than lower-dose FK506 alone at 48 hours, 72 hours, and 7 days after LT. In terms of mechanism, FK506 induced autophagy which resulted in significantly reduced apoptosis and maintained proliferative potential. However, autophagy induced by FK506 also lead to high-mobility group box (HMGB) 1 release from nuclei, resulting in hepatocyte injury through triggering of p38 phosphorylation and chemokine release. Iso effectively inhibited the release of HMGB1 and downstream inflammatory cytokines.. Iso could inhibit release of HMGB1 by FK506 and enhance the hepatoprotective effect of FK506 in rat LT. Combining Iso with FK506 would be promising for the patients after LT.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Autophagy; Cell Line; Hepatocytes; HMGB1 Protein; Humans; Immunosuppressive Agents; Liver; Liver Function Tests; Liver Transplantation; Magnesium; Male; Rats; Rats, Inbred Lew; Reperfusion Injury; Saponins; Tacrolimus; Triterpenes

Adenosine signaling via A1 receptor (A1R) and A2A receptor (A2AR) has shown promise in revealing potential targets for neuroprotection in cerebral ischemia. We recently showed a novel mechanism by which A1R activation with N(6)-cyclopentyl adenosine (CPA) induced GluA1 and GluA2 AMPA receptor (AMPAR) endocytosis and adenosine-induced persistent synaptic depression (APSD) in rat hippocampus. This study further investigates the mechanism of A1R-mediated AMPAR internalization and hippocampal slice neuronal damage through activation of protein phosphatase 1 (PP1), 2A (PP2A), and 2B (PP2B) using electrophysiological, biochemical and imaging techniques. Following prolonged A1R activation, GluA2 internalization was selectively blocked by PP2A inhibitors (okadaic acid and fostriecin), whereas inhibitors of PP2A, PP1 (tautomycetin), and PP2B (FK506) all prevented GluA1 internalization. Additionally, GluA1 phosphorylation at Ser831 and Ser845 was reduced after prolonged A1R activation in hippocampal slices. PP2A inhibitors nullified A1R-mediated downregulation of pSer845-GluA1, while PP1 and PP2B inhibitors prevented pSer831-GluA1 downregulation. Each protein phosphatase inhibitor also blunted CPA-induced synaptic depression and APSD. We then tested whether A1R-mediated changes in AMPAR trafficking and APSD contribute to hypoxia-induced neuronal injury. Hypoxia (20 min) induced A1R-mediated internalization of both AMPAR subunits, and subsequent normoxic reperfusion (45 min) increased GluA1 but persistently reduced GluA2 surface expression. Neuronal damage after hypoxia-reperfusion injury was significantly blunted by pre-incubation with the above protein phosphatase inhibitors. Together, these data suggest that A1R-mediated protein phosphatase activation causes persistent synaptic depression by downregulating GluA2-containing AMPARs; this previously undefined role of A1R stimulation in hippocampal neuronal damage represents a novel therapeutic target in cerebral ischemic damage.

Topics: Animals; Excitatory Postsynaptic Potentials; Furans; Hippocampus; Lipids; Male; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Protein Transport; Pyrones; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, AMPA; Reperfusion Injury; Synaptic Transmission; Tacrolimus

Clinical intestinal transplantation (Int-Tx) is associated with some problems such as rejection, infection, graft-versus-host disease, and ischemia-reperfusion injury (IRI). To determine mechanisms of rejection as well as to develop treatment strategies for Int-Tx, this study was designed to establish both heterotopic and orthotropic Int-Tx models using major histocompatibility antigen complex (MHC) inbred CLAWN miniature swine.. Eleven CLAWN miniature swine received MHC matched but minor antigen mismatched allogenic intestinal grafts. Four animals received intestinal grafts heterotopically and kept host intestine intact. The remaining 7 animals received intestinal grafts orthotopically and resected host small intestine. Continuous infusion of tacrolimus was given from day 0 for 12 days.. Heterotopically transplanted small intestine were well perfused after revascularization; however, grafts easily underwent ischemic changes during or soon after abdomen closure due to oppression of the grafts in the limited abdominal space. In contrast, all of 7 orthotopically transplanted intestinal grafts in which recipients' small intestine was removed from the jejunum to the ileum had no signs of severe ischemia associated with compartment syndrome. Elevation of the serum concentration of inflammatory cytokines and the progression of lethal acidosis seen in recipients of heterotipic transplantation were markedly less in the case of orthotopic transplantation. Two recipients survived more than 30 days, and 1 long-term survivor showed no evidence of rejection at day 90 despite the fact that tacrolimus was stopped at day 12.. In this study, we demonstrated the establishment of a clinically relevant orthotopic Int-Tx model with long survival in MHC inbred CLAWN miniature swine. We believe that this unique MHC inbred swine Int-Tx model is useful for developing treatment strategies for clinical Int-Tx.

Topics: Animals; Disease Models, Animal; Graft Rejection; Graft vs Host Disease; Ileum; Immunosuppressive Agents; Infusions, Intravenous; Intestine, Small; Jejunum; Major Histocompatibility Complex; Reperfusion Injury; Swine; Swine, Miniature; Tacrolimus; Transplantation, Heterotopic; Transplantation, Homologous

It is well known that hippocampus has a pivotal role in learning, formation and consolidation of memory. Global cerebral ischemia causes severe damage to pyramidal neurons of the CA1 region and usually results in residual neurological deficits following a recovery from ischemia. Scientists investigate to find the molecular mechanism of apoptosis and to use this cell death for clinical treatment.. In this investigation, we evaluated the molecular mechanism of FK-506 in apoptosis using gene expression quantification of BAX and BCL-2 genes in hippocampus following global ischemic/reperfusion.. In the present experimental study, adult male Wistar rats were obtained and housed under standard conditions. Each experimental group consisted of five rats and was equally distributed in the normal control, ischemia/reperfusion, ischemia/reperfusion followed by FK-506. Global ischemia was induced for animals in ischemia and drug groups. In the drug group, moreover, two doses of FK-506 were injected as IV injection and intra-peritoneal (IP) injection after 48 h. Then, hippocampus tissue was removed. Consequently, RNA isolated, cDNA was synthesized and Real-Time PCR was performed. Finally, the obtained data was analyzed statistically (p<0.05).. The quantitative results showed the BAX expression ratio increased approximately 3-times in ischemia/reperfusion (3.11 ± 0.28) group compared to the untreated (NR) and the drug group (p<0.001). The statistical analysis showed a significant difference for BCL-2 expression between the experimental groups (p<0.001). The mRNA level of BCL-2 decreased in the ischemia/reperfusion group, while it was without alteration in the other groups.. The results showed that global cerebral ischemia/reperfusion induced BAX as pro-apoptotic gene and tacrolimus a neuroprotective drug inhibited BAX gene expression and induced BCL-2 gene expression as anti-apoptotic gene (Tab. 2, Fig. 3, Ref. 21).

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; CA1 Region, Hippocampal; Male; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tacrolimus; Transcriptome

Purposes; FK506 (strong immunosuppressive agent) was investigated experimentally whether to protect the hepatic IRI. Methods; Warm ischemic experiment using pigs and rats were performed and examined whether FK506 is effective. Results; The results obtained are as follows. 1. Warm ischemia allowed time of the pigs without FK506 was 150 minutes, but as for that of FK506 group, the extension of 30 minutes was got in 180 minutes. 2. Biliary excretion rate of BSP after reperfusion were better in the group of 180 minutes ischemia with FK506 than in without FK506 group. 3. Chemiluminescence intensity in the peripheral neutrophils and adhered and infiltrated leukocytes in the liver were suppressed markedly by FK506. 4. The vascular endothelium with the scanning electron microscope was relatively preserved in the FK506 group comparing to the placebo group on 30 minutes after reperfusion. 5. Stress gastric ulcer was controlled and myeloperoxidase activity in the gastric mucosa was suppressed by FK506. Conclusion; Based on the results of theses experiments, it was suggested that FK506 has a protective effect on IRI by suppressing: the impairment of sinusoidal endothelial cells; the activation of KCs; the disturbance of micro-circulation; oxidative stress; inflammation; and the accumulation of leukocytes. J. Med. Invest. 63: 262-269, August, 2016.

Topics: Animals; CD4-Positive T-Lymphocytes; Female; Kupffer Cells; Liver; Male; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Swine; Tacrolimus

To evaluate renal histological changes and renal function in single kidney rats submitted to renal ischemia-reperfusion and to immunosuppression with tacrolimus and mycophenolate-mofetil.. Experimental study with 80 Wistar rats distributed into control, Sham and six other groups treated with immunosuppressive drugs. Animals undergoing surgery, right nephrectomy and left renal clamping, killed on the 14th day and analyzed for renal histology, urea and creatinine.. The group receiving tacrolimus at higher doses (T3) showed renal histological lesions indicative of early nephrotoxicity, and significant increase in urea and creatinine. The group M (mycophenolate-mofetil alone) and the group M2 (mycophenolate-mofetil combined with half the usual dose of tacrolimus) presented a slight rise in serum urea. The groups using mycophenolate-mofetil alone or combined with tacrolimus showed creatinine levels similar to that of the group T3.. Histologically, the association of injury by ischemia-reperfusion with the use of tacrolimus or mycophenolate-mofetil alone demonstrated a higher rate of renal changes typical of early nephrotoxicity. In laboratory, the combination of injury by ischemia-reperfusion with tacrolimus at higher doses proved to be nephrotoxic.

Topics: Animals; Calcineurin Inhibitors; Creatinine; Immunosuppression Therapy; Immunosuppressive Agents; Ischemia; Kidney; Kidney Diseases; Male; Mycophenolic Acid; Nephrons; Random Allocation; Rats, Wistar; Reperfusion Injury; Tacrolimus; Time Factors; Urea

In the present study, the effects of tacrolimus on the activity and expression of tissue factor (TF) were investigated in the ovarian ischemia-reperfusion induced injury in rats. Twenty-eight female rats (8-12 weeks, 300-350 g) were divided into four groups: control, ischemia-reperfusion (IR), tacrolimus treated before ischemia (TBI), and tacrolimus treated before reperfusion (TBR) groups (n=7/per group). TF activity was measured using Quick's method, whereas TF expression was examined immunohistochemically. TF activity was significantly higher in all treated groups compared with the control group. Strong ovarian TF expression was demonstrated in the IR and TBR groups. Moreover, tacrolimus decreased TF activity in the TBI group compared with the IR group. The decreased activity of TF in the ovarian IR model may prevent IR-related inflammation during transplant procedure.

Topics: Animals; Female; Immunosuppressive Agents; Ovary; Rats; Reperfusion Injury; Tacrolimus; Thromboplastin

Since the proportion of patients given thrombolytic therapy with tissue plasminogen activator (t-PA) is very limited because of the narrow therapeutic window, the development of new therapies for ischemic stroke has been desired. We previously reported that liposomes injected intravenously accumulate in the ischemic region of the brain via disruption of the blood-brain barrier that occurs under cerebral ischemia. In the present study, we investigated the efficacy of a liposomal neuroprotective agent in middle cerebral artery occlusion (MCAO) rats to develop ischemic stroke therapy prior to the recovery of cerebral blood flow. For this purpose, PEGylated liposomes encapsulating FK506 (FK506-liposomes) were prepared and injected intravenously into MCAO rats after a 1-h occlusion. This treatment significantly suppressed the expansion of oxidative stress and brain cell damage. In addition, administration of FK506-liposomes before reperfusion significantly ameliorated motor function deficits of the rats caused by ischemia/reperfusion injury. These findings suggest that FK506-liposomes effectively exerted a neuroprotective effect during ischemic conditions, and that combination therapy with a liposomal neuroprotectant plus t-PA could be a promising therapeutic strategy for ischemic stroke.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Liposomes; Male; Neuroprotective Agents; Oxidative Stress; Polyethylene Glycols; Rats; Rats, Wistar; Reperfusion Injury; Stroke; Tacrolimus

Warm-ischemia-induced injuries might be encountered during renal transplants from cadavers and healthy donors. Toll-like receptors (TLR) in ischemia-reperfusion (I/R) injury are one of the indicators of intracellular injury pathways. The intensity of ischemic injury is directly proportionate to high TLR levels. To minimize the I/R injury, we investigated TLR2 and TLR4 levels on rats, which were pretreated with tacrolimus (FK506) before I/R.. Eight Wistar albino rats in the study group were administered .01 mg/kg intramuscular tacrolimus. Administration to the study group was performed 24 and 1 h before warm ischemia. Eight rats in the control group were injected with 0.1 c.c. of distilled water. Blood samples were collected from the tail veins of all the rats on the first, second and third days. Expression levels of TLR2 and TLR4 genes were analyzed using the polymerase chain reaction method, to determine any significant difference between the control and study groups on the days when blood was taken.. TLR2 (p = 0.045) and TLR4 (p = 0.022) levels in the study group were found to be statistically, and significantly, lower than those in the control group, on the second day following warm-ischemia- and reperfusion-induced injury.. Administration of immunosuppressive drugs to healthy donor rats led to a statistically significant reduction in the expression levels of TLR2 and TLR4 in the early period. In light of the data obtained by this study, we hypothesize that a preoperative therapy on donors might have a role in preventing I/R injury.

Topics: Animals; Gene Expression; Immunosuppressive Agents; Kidney Transplantation; Preoperative Care; Rats; Rats, Wistar; Reperfusion Injury; RNA; Tacrolimus; Toll-Like Receptor 2; Toll-Like Receptor 4

To evaluate the effects of the immunosuppressant tacrolimus as an antioxidant and analyze the histopathologic changes in rat uteri exposed to experimental ischemia-reperfusion (I/R) injury.. Experimental study.. Experimental surgery laboratory in a university.. Twenty-eight female rats exposed to experimentally induced uterine I/R injury.. Group I: control group; group II: uterine I/R injury-induced group; group III: pre-ischemia tacrolimus group; group IV: post-ischemia tacrolimus group.. Uterine tissue malondialdehyde (MDA) level as a marker of lipid peroxidation and glutathione (GSH) level and superoxide dismutase (SOD) and catalase (CAT) activities as markers of tissue antioxidant capacity; histopathologic examination of all uterine rat tissue.. Following aortic I/R injury, MDA levels were significantly increased whereas GSH levels and CAT and SOD activities were found to be decreased compared with control animals. MDA levels were found to recover prominently after the administration of tacrolimus in both groups III and IV. Administration of tacrolimus improved uterine GSH levels and CAT activity in the tacrolimus-treated groups.. Our results indicate that tacrolimus reduces oxidative damage in rat uteri exposed to I/R injury induced by distal abdominal aortic occlusion. Histologic evaluation reveals that tacrolimus attenuates the inflammatory response and protects the tissue damage induced by I/R injury.

Topics: Animals; Antioxidants; Endometritis; Female; Immunosuppressive Agents; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus; Treatment Outcome

The aim of this study was to determine the effect of everolimus and tacrolimus pretreatments on renal morphology and function in a rat ischemia reperfusion (I/R) model. Twenty-eight male Sprague-Dawley rats were randomly assigned to saline + sham operation, saline + I/R (IR), tacrolimus + I/R (TRL + I/R) and everolimus + I/R (ERL + I/R) groups. Saline and active treatments were administered intraperitoneally for seven consecutive days before the surgery. The suprarenal aorta was clamped to achieve warm ischemia, except in the sham group. Right nephrectomy was performed in all animals and histology was examined. Renal function was assessed on post-operative Day 7 by Tc-99m dimercaptosuccinic acid (DMSA) scintigraphy, glomerular filtration rate (GFR) and serum biochemistry. Both everolimus and tacrolimus preserved serum creatinine and blood urea nitrogen levels, but only everolimus preserved GFR (0.74 ± 0.36, 1.20 ± 0.37 and 2.24 ± 0.32 mL/min for I/R, TRL + I/R and ERL + I/R, respectively, P < 0.001). %ID values for sham, I/R, TRL + I/R and ERL + I/R were 55 ± 3, 47 ± 4, 45 ± 6 and 62 ± 7 (P < 0.001). On histologic evaluation, ERL + I/R showed less tubular damage and necrosis than I/R, as well as TRL + I/R. Within the confines of this rat warm ischemia model, everolimus pre-treatment was useful in preserving renal function following I/R injury. The possibility of using everolimus as a pre-conditioning agent for I/R injury in kidney transplantation should be further explored.

Topics: Animals; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Everolimus; Glomerular Filtration Rate; Immunosuppressive Agents; Ischemic Preconditioning; Kidney; Kidney Transplantation; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Tacrolimus; Warm Ischemia

Reactive oxygen species (ROS) have multiple physiological effects that are amount-dependent. ROS are one of the causes of intestinal ischemia-reperfusion (I/R) injury. In this study, we investigated whether the amount of ROS and the degree of intestinal I/R injury affect the expression level of P-glycoprotein (P-gp).. . We used hydrogen peroxide (H2O2) as ROS in in vitro experiments. Intestinal I/R model rats, which were subjected 15-min ischemia (I/R-15), were used in in vivo experiments.. P-gp expression in Caco-2 cells was increased in response to 1 µM of H2O2 but decreased upon exposure to 10 mM of H2O2. We previously reported that P-gp expression is decreased after intestinal I/R with 30-min ischemia (I/R-30), which time a large amount of ROS is generated. I/R-15 induced slightly less mucosal and oxidative injury than did I/R-30. P-gp expression in the jejunum was increased at 1 h after I/R-15, and ileal paracellular permeability was increased. The blood concentration of tacrolimus, a P-gp substrate, was lower during 0-20 min but was higher during 40-90 min post-administration compared with that in the sham-operated rats. P-gp expression in the ileum was decreased at 6 h after I/R-15, due to abnormal localization of P-gp, resulting in a high blood tacrolimus concentration in rats reperfused for 6 h.. ROS multimodally regulate P-gp expression depending on its amount. This is important for understanding the pattern of P-gp expression after intestinal I/R.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caco-2 Cells; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Hydrogen Peroxide; Intestinal Mucosa; Intestines; Male; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Structure-Activity Relationship; Tacrolimus

To evaluate bladder histology in healing and biochemical analysis of rats with single kidney in ischemia/reperfusion, treated with tacrolimus.. Fifty rats randomized into five groups. Three rats died in surgery, 47 rats divided in groups: Control (non-operated, n=10), Sham (operated without drugs, n=8), T1 (operated + tacrolimus 1mg/kg, n=10), T2 (operated + tacrolimus 0.1 mg/kg, n=10), T3 (operated + tacrolimus 10mg/kg, n=9). The surgery was: laparotomy, right nephrectomy, left kidney ischemia/reperfusion, cystotomy followed by bladder suture. After that, rats were submited to gavage daily (Control and Sham with saline solution. T1, T2, T3 with tacrolimus in doses already mentioned). On the 14th day, after death induction, cystectomy was performed and bladder was histologicaly analysed. The serum urea, creatinine and tacrolimus were analysed too.. There was difference in serum tacrolimus in T3 compared to the other groups (p<0.05). There was higher doses of creatinine in T3 group and higher urea in groups with tacrolimus. There were significant differences among all histologic variables comparing groups with and without tacrolimus (p<0.05).. Tacrolimus associated with ischemia/reperfusion is nephrotoxic, suppresses inflammation and seems to delay the healing bladder.

Topics: Animals; Blood Urea Nitrogen; Cicatrix; Creatinine; Immunosuppressive Agents; Ischemia; Kidney; Male; Models, Animal; Nephrectomy; Random Allocation; Rats, Wistar; Reperfusion Injury; Tacrolimus; Urinary Bladder; Wound Healing

To evaluate the impact of a novel oxygenated perfusion approach on rejection after orthotopic liver transplantation (OLT).. Hypothermic oxygenated perfusion (HOPE) was designed to prevent graft failure after OLT. One of the mechanisms is downregulation of Kupffer cells (in situ macrophages). We, therefore, designed experiments to test the effects of HOPE on the immune response in an allogeneic rodent model of nonarterialized OLT.. Livers from Lewis rats were transplanted into Brown Norway rats to induce liver rejection in untreated recipients within 4 weeks. Next, Brown Norway recipients were treated with tacrolimus (1 mg/kg), whereas in a third group, liver grafts from Lewis rats underwent HOPE or deoxygenated machine perfusion for 1 hour before implantation, but recipients received no immunosuppression. In a last step, low-dose tacrolimus treatment (0.3 mg/kg) was assessed with and without HOPE.. Allogeneic OLT without immunosuppression led to death within 3 weeks after nonarterialized OLT due to severe acute rejection. Full-dose tacrolimus prevented rejection, whereas low-dose tacrolimus led to graft fibrosis within 4 weeks. HOPE treatment without immunosuppression also protected from lethal rejection. The combination of low-dose tacrolimus and 1-hour HOPE resulted in 100% survival within 4 weeks without any signs of rejection.. We demonstrate that allograft treatment by HOPE not only protects against preservation injury but also impressively downregulates the immune system, blunting the alloimmune response. Therefore, HOPE may offer many beneficial effects, not only to rescue marginal grafts but also by preventing rejection and the need for immunosuppression.

Topics: Animals; Disease Models, Animal; Down-Regulation; Graft Rejection; Hypothermia, Induced; Kupffer Cells; Liver Transplantation; Oxygen; Perfusion; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate; Tacrolimus

Systemic tacrolimus therapy has been shown to protect against lung ischemia-reperfusion injury in animal models. We sought to investigate on a functional and cellular level if inhaled nanoparticle tacrolimus administered to the donor lung before procurement could similarly attenuate ischemia-reperfusion injury after lung transplant.. An isogenic orthotopic rat model of single left lung transplant was used. Donor animals were pretreated with inhaled tacrolimus (treatment group) or inhaled lactose (controls) before lung procurement. Lung grafts were subjected to 3 hours of cold ischemia followed by 4 hours of reperfusion after graft implantation. Recipient animal arterial blood gas measurement and isograft wet to dry weight ratios were obtained. Macrophage, neutrophil, and T-cell accumulation and activation in lung isografts, including γδ T-cell, T-helper, and cytotoxic T-cell subtypes were analyzed by flow cytometry. Tacrolimus levels were measured in the lung isograft using liquid chromatography/mass spectrometry. Isograft cytokine levels were measured with commercial enzyme-linked immunosorbent assay and microbead array kits.. Oxygenation in treatment group animals was significantly higher than in controls. The presence of macrophages, neutrophils, and all T-cell subtypes in the isografts as well as isograft levels of inflammatory cytokines were all less in the treatment group versus controls, although no single variable achieved statistical significance.. Inhaled nanoparticle tacrolimus treatment of lung donors is associated with an attenuation of ischemia-reperfusion injury on a functional and cellular level in lung transplant.

Topics: Administration, Inhalation; Animals; Blood Gas Analysis; Chemotaxis; Chromatography, High Pressure Liquid; Cytokines; Disease Models, Animal; Flow Cytometry; Immunosuppressive Agents; Lung Transplantation; Macrophages; Male; Nanoparticles; Neutrophil Infiltration; Rats; Rats, Inbred F344; Reperfusion Injury; T-Lymphocyte Subsets; Tacrolimus; Tandem Mass Spectrometry

The effects of tacrolimus postconditioning on protein-serine-threonine kinases (Akt) phosphorylation and apoptotic cell death in rats after spinal cord ischemia-reperfusion injury were investigated. Ninety male SD rats were randomly divided into sham operation group, ischemia-reperfusion group and tacrolimus postconditioning group. The model of spinal cord ischemia was established by means of catheterization through femoral artery and balloon dilatation. The spinal cord was reperfused 20 min after ischemia via removing saline out of balloon. The corresponding spinal cord segments were excised and determined for Akt activity in spinal cord tissue by using Western blotting at 5, 15, and 60 min after reperfusion respectively. Spinal cord tissue sections were stained immunohistochemically for detection of the phosphorylated Akt expression at 15 min after reperfusion. Flow cytometry was applied to assess apoptosis of neural cells, and dry-wet weights method was employed to measure water content in spinal cord tissue at 24 h after reperfusion. The results showed that the activities of Akt in tarcolimus postconditioning group were significantly higher than those in ischemia-reperfusion group at 5, 15, and 60 min after reperfusion (P<0.05, P<0.01). The Akt activities reached the peak at 15 min after reperfusion in ischemia-reperfusion group and tacrolimus postconditioning group. The percentage of apoptotic cells and water content in spinal cord tissue were significantly reduced (P<0.01) in tacrolimus postconditioning group as compared with those in ischemia-reperfusion group at 24 h after reperfusion. It is concluded that tacrolimus post-conditioning can increase Akt activity in spinal cord tissue of rats, inhibit apoptosis of neural cells as well as tissue edema, and thereby alleviate spinal cord ischemia-reperfusion injury.

Topics: Animals; Apoptosis; Immunosuppressive Agents; Male; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Spinal Cord; Spinal Cord Ischemia; Tacrolimus; Up-Regulation

To investigate whether protective effect of tacrolimus postconditioning on rats' spinal cord ischemia-reperfusion injury is mediated by up-regulation of activity of endogenous antioxidant enzymes and down-regulation of production of oxygen free radicals.. Ninety male Sprague-Dawley rats were randomly divided into ischemia-reperfusion (IR) group, tacrolimus postconditioning (TP) group and sham operation (SO) group. The model of spinal cord ischemia was prepared by means of catheterization through femoral artery and balloon dilatation. IR group underwent reperfusion 20 min after spinal cord ischemia. TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery at the onset of reperfusion. SO group received femoral artery catheterization only. Fluoro spectro photometry was employed to detect the level of reactive oxygen species (ROS) in injured spinal cord segment at 15 minutes after reperfusion. The content of malondialdehyde (MDA) and activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) were determined at 15 minutes, 1, 6, and 24 hours after reperfusion respectively. BBB scale was conducted to evaluate hindlimb motor function at 14 days after reperfusion.. The level of ROS in TP group was significantly lower than that in IR group at 15 minutes after reperfusion. The activity of SOD was significantly higher in TP group than in IR group at all observational time points, while the activities of CAT and GSH-PX were significantly higher in TP group than in IR group at 1 and 6 hours after reperfusion. The content of MDA in TP group was significantly less than that in IR group at all observational time points. The motor function score of TP group was significantly superior to that of IR group at 14 days after reperfusion.. Tacrolimus post conditioning can improve activity of endogenous antioxidant enzymes, decrease production of oxygen free radicals, suppress lipid peroxidation, and thereby promote functional recovery after spinal cord ischemia-reperfusion injury in rats.

Topics: Animals; Disease Models, Animal; Glutathione Peroxidase; Ischemic Postconditioning; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Spinal Cord Ischemia; Superoxide Dismutase; Tacrolimus

FK506 (Tacrolimus) has the potential to decrease cerebral ischemia-reperfusion injury. However, the clinical trial of FK506 as a neuroprotectant failed due to adverse side effects. This present study aimed to conduct the selective delivery of FK506 to damaged regions, while at the same time reducing the dosage of FK506, by using a liposomal drug delivery system. First, the cytoprotective effect of polyethylene glycol-modified liposomes encapsulating FK506 (FK506-liposomes) on neuron-like pheochromocytoma PC12 cells was examined. FK506-liposomes protected these cells from H2O2-induced toxicity in a dose-dependent manner. Next, we investigated the usefulness of FK506-liposomes in transient middle cerebral artery occlusion (t-MCAO) rats. FK506-liposomes accumulated in the brain parenchyma by passing through the disrupted blood-brain barrier at an early stage after reperfusion had been initiated. Histological analysis showed that FK506-liposomes strongly suppressed neutrophil invasion and apoptotic cell death, events that lead to a poor stroke outcome. Corresponding to these results, a single injection of FK506-liposomes at a low dosage significantly reduced cerebral cell death and ameliorated motor function deficits in t-MCAO rats. These results suggest that liposomalization of FK506 could reduce the administration dose by enhancing the therapeutic efficacy; hence, FK506-liposomes should be a promising neuroprotectant after cerebral stroke.

Topics: Animals; Cells, Cultured; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Liposomes; Male; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus

Ischemia and reperfusion injury to the liver remains an important clinical problem during liver transplantation and the protection of myocardial function is critical for patients suffering liver transplantation. In the present study, we evaluated the effect of tacrolimus on injured myocardium and mitochondria after total hepatic ischemia-reperfusion (THIR).. We utilized the THIR model to mimic the liver transplantation and evaluate the myocardial injury after THIR. Male Wistar rats were divided into four groups: control (sham operated); THIR; THIR+tacrolimus (intraperitoneally, 0.1 mg/kg), and tacrolimus alone group.. THIR indeed damaged the myocardial and mitochondrial function, including increasing the level of MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH), decreasing mitochondrial transmembrane potential (MTP), mitochondrial respiratory and phosphorylating activities (P < 0.05). After administration of tacrolimus, myocardial injuries were significantly attenuated (P < 0.05).. THIR injury was associated with myocardial injury, perhaps resulting from mitochondrial function alterations. The inhibition of calcineurin and the improvement of mitochondrial respiration and antioxidant capacity maybe contributed to the protection of myocardial mitochondria function after THIR.

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Creatine Kinase, MB Form; Heart Injuries; Immunosuppressive Agents; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Models, Animal; Myocardium; Rats; Rats, Wistar; Regional Blood Flow; Reperfusion Injury; Sodium-Potassium-Exchanging ATPase; Tacrolimus

To characterize the immunosuppressant tacrolimus as a protective antioxidant in rat liver transplantation.. Livers of male Lewis rats underwent 24 h of hypothermic preservation in UW solution and were rinsed with tacrolimus or placebo directly before transplantation. Markers of liver injury, such as enzymes and bile flow, were determined during a 2 h reperfusion period. Concentrations of reduced (GSH) and oxidized (GSSG) glutathione were analyzed in plasma, bile, and liver tissue for estimation of oxidant stress caused by reactive oxygen species (ROS).. Administration of tacrolimus (10 ng/mL) resulted in decreased ALT plasma levels (1740 ± 1169 U/l versus 3691 ± 1144 U/l; P < 0.05) at 2 h of reperfusion. While endogenous intracellular GSH concentrations remained unchanged, GSSG, the oxidation product of GSH, was markedly decreased at 2 h of reperfusion in preconditioned livers (47.0 ± 10.4 nm/g versus 71.8 ± 30.6 nm/g; P < 0.05). Correspondingly, GSSG bile concentrations (0.19 ± 0.04 mM versus 0.13 ± 0.04 mM; P < 0.05) as well as plasma GSSG levels (2.4 ± 0.3 mM versus 1.4 ± 0.2 mM; P < 0.05) were significantly increased upon reperfusion. These findings suggest that tacrolimus impacts post-ischemic GSH metabolism when administered as a rinse solution for liver allografts through an unknown pathway.. Hepatocellular injury following transplantation was significantly decreased by preconditioning with tacrolimus. One possible mechanism of action is the detoxification of ROS through the preservation of cytosolic and extracellular GSH/GSSG ratios.

Topics: Animals; Antioxidants; Dose-Response Relationship, Drug; Glutathione; Glutathione Disulfide; Homeostasis; Immunosuppressive Agents; Ischemic Preconditioning; Liver; Liver Transplantation; Male; Models, Animal; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Reperfusion Injury; Tacrolimus; Transplantation, Homologous

Reperfusion injury remains one of the major problems in transplantation. Repair from ischaemic acute renal failure (ARF) involves stimulation of tubular epithelial cell proliferation. The aim of this exploratory study was to evaluate the effects of preconditioning donor animals with rapamycin and tacrolimus to prevent ischaemia-reperfusion (I/R) injury. Twelve hours before nephrectomy, the donor animals received immunosuppressive drugs. The animals were divided into four groups, as follows: group 1 control: no treatment; group 2: rapamycin (2 mg/kg); group 3 FK506 (0, 3 mg/kg); and group 4: FK506 (0, 3 mg/kg) plus rapamycin (2 mg/kg). The left kidney was removed and after 3 h of cold ischaemia, the graft was transplanted. Twenty-four hours after transplant, the kidney was recovered for histological analysis and cytokine expression. Preconditioning treatment with rapamycin or tacrolimus significantly reduced blood urea nitrogen and creatinine compared with control [blood urea nitrogen (BUN): P < 0·001 versus control and creatinine: P < 0·001 versus control]. A further decrease was observed when rapamycin was combined with tacrolimus. Acute tubular necrosis was decreased significantly in donors treated with immunosuppressants compared with the control group (P < 0·001 versus control). Moreover, the number of apoptotic nuclei in the control group was higher compared with the treated groups (P < 0·001 versus control). Surprisingly, only rapamycin preconditioning treatment increased anti-apoptotic Bcl2 levels (P < 0·001). Finally, inflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, showed lower levels in the graft of those animals that had been pretreated with rapamycin or tacrolimus. This exploratory study demonstrates that preconditioning donor animals with rapamycin or tacrolimus improves clinical outcomes and reduce necrosis and apoptosis in kidney I/R injury.

Topics: Animals; Apoptosis; Blood Urea Nitrogen; Complement C3; Creatinine; Cytokines; Drug Evaluation; Drug Synergism; Drug Therapy, Combination; Immunosuppressive Agents; Kidney Transplantation; Kidney Tubular Necrosis, Acute; Male; Postoperative Complications; Premedication; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; Tacrolimus

The immunosuppressant tacrolimus (FK506) has improved pancreas allograft survival compared with cyclosporin A (CsA), possibly because of reduced acute pancreatitis following ischemia-reperfusion injury. Ion permeabilities in zymogen granule (ZG) membranes, including a KCNQ1 K channel, promote hormone-stimulated enzyme secretion. We investigated whether a differential modulation of ZG and lysosomal ion permeabilities and enzyme secretion by CsA/FK506 contributes to pancreatitis.. Rat ZGs and lysosomes were isolated by gradient centrifugation, ion permeabilities assayed by osmotic lysis, and single-channel currents recorded in a planar lipid bilayer. Amylase release was measured in permeabilized acini and lysosomal cathepsin B release detected by immunoblotting.. CsA (1-10 μM), but not FK506, enhanced ZGs osmotic lysis by selectively increasing K permeability up to 5-fold. Zymogen granule membrane K channels showed ∼2-fold increased single-channel open probability with CsA only. Cyclosporin A selectively increased basal (∼2-fold), but not cholecystokinin-octapeptide (1 nM)-induced amylase secretion in K medium only. Cyclosporin A (5 μM), but not FK506, increased cathepsin B release from lysosomes.. Cyclosporin A selectively opens the ZG K channel and induces cathepsin B release from lysosomes, which cause increased in situ lysis of ZGs and may aggravate or fuel acute allograft pancreatitis following hypoxia-reperfusion injury.

Topics: Acinar Cells; Amylases; Animals; Biomarkers; Cathepsin B; Cyclosporine; Immunosuppressive Agents; Lysosomes; Male; Osmosis; Pancreas; Pancreas Transplantation; Pancreatitis, Graft; Permeability; Potassium Channels; Rats; Rats, Wistar; Reperfusion Injury; Secretory Vesicles; Tacrolimus

The ischemia-reperfusion injury (IRI) remains a major problem in transplantation. The objective of this study was to evaluate the effects of preconditioning a donor group with rapamycin and another donor group with tacrolimus to prevent IRI. Twelve hours before nephrectomy, donor Wistar rats received immunosuppressive drugs. The sample was divided into four experimental groups: a sham group, an untreated control group, a group treated with rapamycin (2 mg/kg) and a group treated with tacrolimus (0.3 mg/kg). Left kidneys were removed and, after three hours of cold ischemia, grafts were transplanted. Twenty-four hours later, the transplanted organs were recovered for histological analysis and evaluation of cytokine expression. The pre-conditioning treatment with rapamycin or tacrolimus significantly reduced donor blood urea nitrogen and creatinine levels compared with control group (BUN: p < 0.001 vs. control and creatinine: p < 0.001 vs. control). Acute tubular necrosis was significantly lower in donors treated with immunosuppressant drugs compared with the control group (p < 0.001). Finally, inflammatory cytokines such as TNF-a, IL-6 and rIL-21 showed lower levels in the graft of pre-treated animals. This exploratory experimental study shows that preconditioning donors with rapamycin and tacrolimus in different groups improves clinical outcome and pathology in recipients and reduces in situ pro-inflammatory cytokines associated with Th17 differentiation, creating a favorable environment for the differentiation of regulatory T cells (Tregs).

Topics: Animals; Cytokines; Disease Models, Animal; Immunosuppression Therapy; Immunosuppressive Agents; Inflammation; Inflammation Mediators; Kidney Transplantation; Living Donors; Male; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; Tacrolimus; Transplantation Conditioning; Tumor Necrosis Factor-alpha

Allograft inflammatory factor-1 (AIF-1) plays an important role in immune response and vasculopathy in allografts. The present study investigated activation of AIF-1 in warm and cold ischemia-reperfusion (IR) injury of rat liver, and the potential inhibitory effect of FK506.. We used warm IR injury, orthotopic transplantation, and allograft rejection models in this study. We assessed expression of AIF-1 mRNA and protein, as well as its inducers interferon-γ (IFN-γ) and interleukin-1β (IL-1β). The potential inhibitory effect of FK506 on AIF-1 in a rat macrophage cell line and in these three models was also assessed.. AIF-1 mRNA and protein, as well as its inducers IFN-γ and IL-1β, were significantly increased in the warm IR injury, orthotopic transplantation, and allograft rejection models. Real-time RT-PCR and Western blotting showed that pretreatment with low-dose FK506 partially inhibited AIF-1 activation as well as its inducers (IFN-γ and IL-1β) in these three models. Western blotting showed that IFN-γ and IL-1β activated AIF-1 in a macrophage cell line, but pretreatment with FK506 did not inhibit AIF-1 activation in vitro. Hematoxylin and eosin staining showed that edema and necrosis in the liver, as well as alanine aminotransferase (ALT) in serum, of these three groups was reduced after FK506 pretreatment.. AIF-1 was activated in warm and cold IR injury, and pretreatment with low-dose FK506 partly inhibited AIF-1 activation and reduced warm and cold IR injury.

Topics: Alanine Transaminase; Animals; Calcium-Binding Proteins; Cell Line; Immunosuppressive Agents; Liver; Male; Microfilament Proteins; Rats; Rats, Inbred Lew; Reperfusion Injury; Tacrolimus; Up-Regulation

Ischemia reperfusion injury (IRI) is one of the risk factors for delayed graft function, acute rejection and long term allograft survival after kidney transplantation. IRI is an independent antigen inflammatory process that produces tissue damage. Our objective was to study the impact of immunosuppressive treatment (IS) on IRI applying only one dose of IS before orthotopic kidney autotransplantation.. Twenty-four rats allocated in four groups were studied. One group served as control (G1: autotransplanted rats without IS) and the rest received IS 12 h before kidney autotransplantation (G2: Rapamycin, G3: Mycophenolate mofetil and G4: Tacrolimus).. Improved renal function and systemic inflammatory response were found among IS groups compared to the control group (Delta Urea p<0.0001; Delta Creatinine p<0.0001; Delta C3 p<0.001). The number of apoptotic nuclei in renal medulla in G1 was higher than in IS groups (p<0.0001). Tubular damage was less severe in IS groups respecting G1 (p<0.001). C3, TNF-α and IL-6 expression in kidney samples was reduced when IS was used compared to the control group. No differences were observed among the different immunosuppressive drugs tested. However, Heme oxygenase-1(HO-1) was increased only in Rapamycin treatment.. These data suggest that the use of IS administered before transplant attenuates the IRI process after kidney transplantation in an animal model.

Topics: Animals; Apoptosis; Complement C3; Creatinine; Graft Survival; Heme Oxygenase-1; Immunosuppressive Agents; Interleukin-6; Kidney Function Tests; Kidney Transplantation; Kidney Tubules; Male; Mycophenolic Acid; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; Tacrolimus; Transplantation, Autologous; Urea

Ischemia-reperfusion (I/R) is present at various degrees in kidney transplants. I/R plays a major role in early function and long-term survival of renal allograft. The purpose of our study was to determine if immunosuppressants modulate I/R in a model that separates I/R from all immune responses.. Sprague-Dawley rats with monolateral renal I/R received daily cyclosporine (A), tacrolimus (B), sirolimus (C) or saline (D). Sham-operated rats received saline (E). After 30 days, glomerular filtration rate for each kidney was measured by inulin clearance. Kidney injury was examined, and TGF-β, fibronectin and metalloproteases were evaluated by real-time PCR, Western blot and zymography.. Sirolimus, but not cyclosporine and tacrolimus, prevented a glomerular filtration rate decrease in I/R kidneys (403 ± 303 vs. 1,006 ± 484 μl/min, p < 0.05; 126 ± 170 vs. 567 ± 374 μl/min, p < 0.05; 633 ± 293 vs. 786 ± 255; A, B and C group, respectively, I/R vs. contralateral kidneys). Sirolimus reduced ED-1+ cell infiltrate, interstitial fibrosis and intimal thickening of small vessels observed in I/R kidneys of controls and calcineurin inhibitor-treated rats. Tacrolimus and cyclosporine increased fibronectin and TGF-β expression and matrix deposition. Only sirolimus increased metalloprotease activity.. Sirolimus but not calcineurin inhibitors prevented I/R-induced kidney injury.

Topics: Animals; Cyclosporine; Glomerular Filtration Rate; Immunosuppressive Agents; Kidney; Kidney Diseases; Kidney Function Tests; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Tacrolimus

The protein phosphatase 2B inhibitor, FK506, is an immunomodulatory polypeptide that has neuroprotective properties, the mechanisms of which have not been elucidated. A possible mechanism may be phosphorylation-mediated regulation of glutamate transporter activity. In the present study, we investigated the effect of FK506 on glutamate transporter localization and activity in the ischaemic mouse retina. FK506 did not appear to modulate the localization or activity of glutamate transporters under simulated ischaemic conditions. Our present data suggest that the mechanism by which FK506 exerts its neuroprotective action is not attributable to alterations in retinal glutamate transport.

Topics: Amino Acid Transport System X-AG; Animals; Biological Transport; Calcineurin Inhibitors; Female; Glutamic Acid; Immunohistochemistry; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Reperfusion Injury; Retinal Diseases; Tacrolimus; Tetradecanoylphorbol Acetate

Ischemia-reperfusion injury impairs lung transplant outcomes. The transcription factors, activator protein-1, and nuclear factor kappa B, are activated early in reperfusion and drive the development of injury. Thrombin inhibition with hirudin, and calcineurin inhibition with tacrolimus have independently been shown to ameliorate lung ischemia-reperfusion injury by reducing activator protein-1 and nuclear factor kappa B activation, respectively. However, high doses were required to achieve protection using individual agents, raising concerns about potential toxicities. We sought to determine if low-dose combination therapy reduced injury through synergistic inhibition of pretranscriptional signaling events.. Rats were pretreated with either intravenous hirudin or tacrolimus at low doses or high doses, or both at low doses, prior to undergoing left lung ischemia and reperfusion. Lungs were assessed for markers of lung injury, including bronchoalveolar lavage cytokine-chemokine content and transcription factor transactivation of activator protein-1 and nuclear factor kappa B.. High-dose monotherapy with hirudin or tacrolimus reduced lung injury and transactivation of activator protein-1 and nuclear factor kappa B activation, respectively, whereas low-dose monotherapy with either agent did not alter transcription factor activation or lung injury compared with positive controls. Low-dose combination therapy was more protective than high-dose monotherapy with either drug, and correlated with a reduction in activation of both transcription factors and their associated cytokines.. The significant decrease in lung injury severity and transcription factor activation with combined pathway inhibition suggests pretranscriptional signaling redundancy between the calcineurin and thrombin dependent pathways in lung reperfusion injury.

Topics: Animals; Calcineurin Inhibitors; Drug Synergism; Drug Therapy, Combination; Hirudin Therapy; Lung; Rats; Rats, Long-Evans; Reperfusion Injury; Tacrolimus; Thrombin

Topics: Animals; Calcineurin Inhibitors; Drug Therapy, Combination; Hirudin Therapy; Lung; Rats; Reperfusion Injury; Tacrolimus; Thrombin

Ischemia/reperfusion injury and delayed graft function (DGF) following organ transplantation adversely affect graft function and survival. A large animal model has not been characterized. We developed a pig kidney allograft model of DGF and evaluated the cytoprotective effects of inhaled carbon monoxide (CO). We demonstrate that donor warm ischemia time is a critical determinant of DGF as evidenced by a transient (4-6 days) increase in serum creatinine and blood urea nitrogen following transplantation before returning to baseline. CO administered to recipients intraoperatively for 1 h restored kidney function more rapidly versus air-treated controls. CO reduced acute tubular necrosis, apoptosis, tissue factor expression and P-selectin expression and enhanced proliferative repair as measured by phosphorylation of retinol binding protein and histone H3. Gene microarray analyses with confirmatory PCR of biopsy specimens showed that CO blocked proinflammatory gene expression of MCP-1 and heat shock proteins. In vitro in pig renal epithelial cells, CO blocks anoxia-reoxygenation-induced cell death while promoting proliferation. This large animal model of DGF can be utilized for testing therapeutic strategies to reduce or prevent DGF in humans. The efficacy of CO on improving graft function posttransplant validates the model and offers a potentially important therapeutic strategy to improve transplant outcomes.

Topics: Animals; Carbon Monoxide; Cell Death; Cell Proliferation; Delayed Graft Function; Female; Gene Expression Profiling; Graft Rejection; Kidney; Kidney Transplantation; Kidney Tubular Necrosis, Acute; Reperfusion Injury; Swine; Tacrolimus

The postischemic intestine liberates pro-inflammatory mediators (cytokines, lipopolysaccharide [LPS], free radicals) proportional with the local injury that may trigger a systemic inflammatory response and multi-system organ failure. Previously, intestines from donors receiving Tacrolimus revealed improved morphology and abrogated nuclear factor kappa B (NF-kappaB) activation. Because of its pivotal role in inflammation, we investigated if NF-kappaB intragraft inhibition influences the posttransplant inflammatory response and remote organ injury.. Donor Sprague Dawley rats received tacrolimus (0.3 mg/kg) or saline i.v. 6 h before graft harvest. The intestines were preserved for 3 h and then transplanted heterotopically. Hepatic microcirculation was assessed at 20 min, 6 h, 12 h, or 24 h post-reperfusion (postR) using laser-Doppler flowmetry (n = 10/group). Blood pressure measurements and liver sampling were performed at 6, 12, or 24 h postR. Blood samples were obtained at 1, 3, 6, 12, and 24 h postR. Hepatic intercellular adhesion molecule 1 (ICAM-1) expression, caspase-3 and -9 activity, and circulating tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and LPS were studied.. Pretreated graft (PG) recipients had superior cardiovascular parameters at 6 and 12 h postR, while liver perfusion was similar between groups at all time points. Recipients of PG had lower transaminase levels and ICAM-1 liver expression. Liver caspase 3 and 9 activity were similar at 6 and 12 h but increased at 24 h in both groups. At every time point, circulating tumor necrosis factor alph, IL-1beta, and IL-6 were lower in animals receiving PG. LPS was found increased only at the last time point.. Transplantation of tacrolimus-pretreated intestines triggered a milder inflammatory response and decreased liver injury early posttransplantation compared with untreated grafts. Cytokines, but not neutrophils, hypoperfusion, or LPS may underlie the dysfunction.

Topics: Animals; Caspase 3; Caspase 9; Cytokines; Immunosuppressive Agents; Interleukin-1beta; Interleukin-6; Intestines; Ischemic Preconditioning; Lipopolysaccharides; Liver; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus; Tumor Necrosis Factor-alpha

The protective effects of immunosuppressants against ischemia/reperfusion (I/R) injury have been attributed to their non-specific anti-inflammatory effect. However, these effects may also depend on their effect on T lymphocytes, which are increasingly considered to be key players in I/R. Here, we studied the effects of tacrolimus and sirolimus on lymphocyte subpopulations in an I/R rat model. The animals were treated with tacrolimus, sirolimus or vehicle, before undergoing a 60-min ischemia event of the right hepatic lobe, followed by excision of the remaining liver. After 2 h, I/R rats showed increased mortality, plasma lactate dehydrogenase (LDH) levels, hepatocyte apoptosis, liver histological injury and parenchymal infiltration by neutrophils, macrophages, NK cells and T lymphocytes. Most of the changes were antagonized by both immunosuppressants. Tacrolimus augmented the proportion of cycling cells in I/R rats, whereas sirolimus showed the opposite effect. The increased Th1/Th2 ratio found in I/R livers after 2 h was reverted by immunosuppressants, which also amplified the proportion of CD4(+)CD25(+)Foxp3(+) regulatory T lymphocytes at 24 h. The protective effects of both tacrolimus and sirolimus correlated well with a decreased ratio of proinflammatory to anti-inflammatory T lymphocytes, and with an increase in the Treg proportion. This suggests a new mechanism to explain the known beneficial effect shown by immunosuppressants early after I/R.

Topics: Animals; Apoptosis; Hepatectomy; Immunosuppressive Agents; Liver; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; T-Lymphocytes, Regulatory; Tacrolimus; Th1 Cells; Th2 Cells

Ischemia/reperfusion (I/R) injury is a main cause of primary dysfunction or non-function after liver transplantation (LTx). Recent evidence indicates that an increase in nitric oxide (NO) production after LTx is associated with I/R injury. The aim of this study was to demonstrate that low-dose FK506 in combination with aminoguanidine (AGH), which leads to a reduction of NO levels, has a protective effect by reducing I/R associated injury after LTx. Fortyone DA-(RT1av1) rats served as donors and recipients for syngenic orthotopic arterialised LTx. They were divided into 4 groups: controls without pre-/treatment (I), pre-/treatment with high-dose FK506 (II), pre-/treatment with AGH only (III), and pre-/treatment with low-dose FK506 in combination with AGH (IV). After LTx the laboratory parameters and liver biopsy were performed. The levels of transaminase (ALT) in groups I, II and III were significantly higher on day 3 after LTx compared to group IV (p = 0.001, p = 0.001, p = 0.000). In group IV the I/R-associated liver necrosis rate was reduced significantly. Our results demonstrated that a combined dual pharmacological pretreatment (group IV) reduced I/R injury of the graft after LTx in a rat model.

Topics: Animals; Dose-Response Relationship, Drug; Drug Therapy, Combination; Guanidines; Liver; Liver Transplantation; Nitric Oxide; Rats; Rats, Inbred Strains; Reperfusion Injury; Tacrolimus; Transplantation Conditioning

Topics: Adrenergic alpha-Antagonists; Animals; Dogs; Extravascular Lung Water; Graft Survival; Immunosuppressive Agents; Lung Transplantation; Phentolamine; Pulmonary Alveoli; Pulmonary Edema; Reperfusion Injury; Tacrolimus

To investigate the effects of a novel Leukotriene B(4) receptor antagonist and/or tacrolimus on ischemia-reperfusion in a rat liver model.. Male Lewis rats were pretreated with ONO-4057 (100 mg/kg) and/or tacrolimus (1 mg/kg) orally, and divided into four experimental groups; group 1 (control), group 2 (ONO-4057), group 3 (tacrolimus), group 4 (ONO-4057 + tacrolimus).. There was a tendency for long survival in the groups treated with tacrolimus alone and ONO-4057 plus tacrolimus. Post-reperfusion serum aspartate aminotransferase levels decreased more significantly in ONO-4057 plus tacrolimus group (P < 0.01), than in the tacrolimus alone group (P < 0.05), compared to controls.. This study demonstrated that pretreatment with ONO-4057 in combination with tacrolimus produced additive effects in a rat model of liver ischemia-reperfusion injury.

Topics: Animals; Immunosuppressive Agents; Liver; Male; Peroxidase; Phenylpropionates; Rats; Rats, Inbred Lew; Reperfusion Injury; Tacrolimus

Tacrolimus (FK506) is widely used as an immunosuppressive drug in small bowel transplantation. However, its precise effects on the vascular tone of the transplanted organ have not been studied. This study aimed to clarify the effects of FK506 on the porcine mesenteric artery.. The effects of FK506 on the changes in cytosolic Ca(2+) concentration ([Ca(2+)]i) and force using fura-2 fluorometry were investigated in mesenteric arterial strips of the porcine small intestine. The effects of FK506 on the activity of voltage-dependent Ca(2+) channels and receptor-operated Ca(2+) channels using high K(+) (118 mmol/L K(+)) depolarization and thromboxane A(2) analog (U46619) stimulation were also examined.. FK506 inhibited the force development induced by 118 mmol/L K(+) depolarization and 1 micromol/L U46619 stimulation in a concentration-dependent manner. The extent of inhibition of this contraction was greater than that of the K(+)-induced contraction, and its inhibitory potency was about 10-fold. FK506 (10 micromol/L) inhibited the increases in [Ca(2+)]i (24.9% +/- 7.4%) and the force development (52.0% +/- 5.6%) induced by 1 micromol/L U46619, respectively.. FK506 induces arterial relaxation by decreasing [Ca(2+)]i. Pretreatment of a graft with FK506 may reduce the risk of vasospasm, ischemia-reperfusion injury, and thrombosis in small bowel transplantation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arteries; Calcium; Calcium Channels; Cytosol; Dose-Response Relationship, Drug; Fluorometry; Fura-2; Immunosuppressive Agents; Intestine, Small; Mesenteric Arteries; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Reperfusion Injury; Swine; Tacrolimus; Vasoconstrictor Agents; Vasodilation

Cyclosporine A has been shown to detrimentally affect post-transplantation vascular tone. Tacrolimus (FK506), an immunosuppressant whose mechanism of action is similar to that of cyclosporine A, is more potent in vitro and has a reportedly high level of safety. The effects of tacrolimus on hepatic vasculature and metabolism in isolated, dually perfused (through both the hepatic artery and porta) rat liver under normal conditions and again in association with a state of hypoperfusion followed by reperfusion, imitating the liver's clinical state during organ transplant, were investigated.. Three groups were perfused normally with Krebs-Henseleit solution and three groups were hypoperfused (75% flow reduction) for two hours. Saline, tacrolimus 4 ng/ml, or tacrolimus 40 ng/ml was injected in three normally perfused and three hypoperfused liver groups to determine drug effects under normal conditions and in low-flow-reflow state. Non-radioactive microspheres were later administered to all livers via the artery to assess microcirculatory patency.. Tacrolimus did not affect the normally perfused livers. Liver hypoperfusion without treatment (saline injection) caused wet-dry weight ratio increase, abnormal increases in hepatic artery pressure and resistance values, and non-physiologically low oxygen extraction during reperfusion. Hypoperfusion + tacrolimus 4 or 40 ng/ml yielded values closer to those of the normally perfused livers. Finally, more microspheres were trapped in the hypoperfused+saline-treated liver circulation than in the normally perfused or hypoperfused+tacrolimus 4 or 40 ng/ml livers.. Tacrolimus appears to be nontoxic in the isolated liver and to mitigate microcirculatory derangement associated with low-flow-reflow states.

Topics: Animals; Immunosuppressive Agents; In Vitro Techniques; Liver; Liver Transplantation; Male; Perfusion; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus

Ischemia/reperfusion injury (IRI) represents the single major antigen-independent factor implicated in pathogenesis of chronic graft dysfunction. Tacrolimus is a calcineurin inhibitor, which has been suggested to be helpful in cyclosporine-related chronic toxicity. Rapamycin has antiproliferative properties that may impair renal regeneration after IRI. Therefore, immunosuppressive drugs might impair renal graft outcome in those organs suffering IRI.. C57B1/6 male mice subjected to 45 minutes of renal pedicle ligation were reperfused for 24 hours. Mice were treated with rapamycin, cyclosporine, or tacrolimus. Blood and renal tissue samples were collected at 24 hours after IRI. Urea levels were measured. Heme Oxygenase 1 (HO-1) gene transcript was amplified by a real-time polymerase chain reaction technique.. Animals treated with cyclosporine and subjected to IRI showed impaired renal function that peaked at 24 hours. Additional pretreatment with rapamycin produced even more impairment of renal function, when compared with controls. However, tacrolimus pretreatment was associated with a better renal outcome. HO-1 expression was upregulated after IRI by 2.6 arbitrary units at 24 hours. Rapamycin showed worse impairment of renal function.. Tacrolimus was not associated with worsening renal function when compared with animals just subjected to IRI. Upregulation of HO-1 may be an attractive approach to limit graft injury.

Topics: Animals; Cyclosporine; Heme Oxygenase-1; Immunosuppressive Agents; Kidney Diseases; Kidney Function Tests; Male; Mice; Mice, Inbred C57BL; Polymerase Chain Reaction; Reperfusion Injury; Sirolimus; Tacrolimus; Transcription, Genetic

FK506 alleviates warm ischemia-reperfusion injury, but it remains unknown if such protection is manifest after cold storage and transplantation. We studied the early outcome after transplantation of intestines from donors pretreated with FK506 compared to grafts from controls treated with saline (154 mM NaCl).. Sprague-Dawley rats received 0.3 mg/kg FK506 or saline intravenously 6 hours before graft retrieval. The small bowel was harvested, stored for 3 hours, and then transplanted heterotopically. Samples were taken after preservation and at 20 minutes, 6 hours, 12 hours, and 24 hours after reperfusion. Heat shock protein 72 (Hsp72) and iintercellular adhesion molecule (ICAM)-1 expression and nuclear factor kappaB (NF-kappaB) activation were assessed via Western blots and eelectrophoretic mobility shift assay (EMSA), respectively. Dissacharidase activity and enterocyte proliferation rate were also studied.. Preservation injury was similar between groups, but pretreated grafts had better morphology already 20 minutes after reperfusion. Control grafts always had thinner mucosa and more PMN infiltration. Hsp72 expression was greater in pretreated grafts. ICAM-1 was absent after harvesting, preservation, and immediately after reperfusion but increased in control grafts at the later time points. Control grafts showed a biphasic NF-kappaB activation pattern, whereas NF-kappaB activation was inhibited effectively in pretreated grafts. Dissacharidase activity decreased during the first 6 hours after reperfusion but recovered within 24 hours in pretreated grafts but not in control grafts. Earlier enterocyte proliferation was observed in pretreated grafts.. FK506 donor pretreatment reduced graft proinflammatory activation and neutrophil inflammation. Pretreated groups revealed a milder reperfusion injury and accelerated morphologic and functional recovery. The mechanisms involved appear to involve Hsp72 upregulation and NF-kappaB inhibition.

Topics: Animals; Blotting, Western; Cell Proliferation; Disaccharidases; Electrophoretic Mobility Shift Assay; Enterocytes; Graft Survival; HSP72 Heat-Shock Proteins; Immunosuppressive Agents; Intercellular Adhesion Molecule-1; Intestine, Small; Male; Neutrophil Infiltration; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus; Tissue Donors; Transplantation, Heterotopic

The mechanisms underlying the neuroprotective effects of the immunosuppressant tacrolimus, observed in vivo, remain unclear. Here we quantify these effects in vitro, and evaluate the potential involvement of the glutamate and/or immunophilin FK506 binding protein 12 kDa in tacrolimus-induced neuroprotection.. Primary cultures of neurons and astrocytes from rat cerebral cortex were subjected to transient oxygen-glucose deprivation. Neuronal injury was evaluated by cell counting after immunostaining experiments, lactate dehydrogenase release and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. The involvement of the immunophilin FK506 binding protein 12 kDa was explored using an anti-FK506 binding protein 12 kDa antibody, (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate and rapamycin. Extracellular glutamate and glutamate uptake were respectively measured by high performance liquid chromatography and l-[3H]glutamate incorporation.. When added during either oxygen-glucose deprivation or reoxygenation, FK506 (50-500 pM) offered significant neuroprotection. During oxygen-glucose deprivation, it was able to reverse the oxygen-glucose deprivation-induced increase in extracellular glutamate and decrease in glutamate uptake and this effect was reversed in the presence of threo-3-methyl glutamate, a specific inhibitor of glutamate transporter-1. Blocking FK506 binding protein 12 kDa inhibited the neuroprotection induced by tacrolimus added during either oxygen-glucose deprivation or reoxygenation. Tacrolimus-induced neuroprotection was also reversed in the presence of rapamycin, an immunosuppressant FK506 binding protein 12 kDa ligand devoid of neuroprotective properties and (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate, a non-immunosuppressant ligand of FK506 binding protein 12 kDa, exerteing neuroprotective effects.. The beneficial effects of tacrolimus during in vitro ischemia/reperfusion seem to indicate the restoration of a glutamate transporter-1-mediated activity and could be mediated by a FK506 binding protein 12 kDa pathway.

Topics: Animals; Astrocytes; Cells, Cultured; Cerebral Cortex; Chromatography, High Pressure Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Glucose; Glutamic Acid; Immunohistochemistry; In Vitro Techniques; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus; Tacrolimus Binding Proteins

Ischemia-reperfusion (I-R) injury is poorly tolerated by fatty livers, most probably secondary to reduced cellular adenosine triphosphate (ATP) levels. We investigated the effectiveness of tacrolimus pretreatment on fatty liver I-R injury in obese Zucker rats. Tacrolimus (0.3 mg/kg, intravenously) was injected 24 hours before a 75-minute ischemic period and rats were sacrificed 6 hours later. Tacrolimus modified the response to I-R observed in obese Zucker rats, when compared to nontreated obese rats: a significant reduction in hepatocyte necrosis was associated with a significant increase in hepatocyte apoptosis. In addition, cell necrosis and apoptosis were significantly and inversely correlated in lean nontreated and treated obese Zucker rats following I-R. Tacrolimus also significantly increased the hepatic ATP levels, reduced in nontreated obese rats, toward values found in lean Zucker rat livers. This protective effect of tacrolimus was further confirmed in vivo by a significantly improved survival following pretreatment with tacrolimus, 24 hours prior to ischemia. In conclusion, in obese Zucker rat livers, tacrolimus pretreatment reversed the I-R injury toward the one found in lean Zucker rats. The correlations between ATP levels and the opposite changes in necrosis and apoptotic pathways strongly suggest a cause-effect relationship between tacrolimus and changes in ATP levels.

Topics: Animals; Biopsy, Needle; Body Weight; Disease Models, Animal; Fatty Liver; Immunohistochemistry; Liver Circulation; Liver Function Tests; Liver Regeneration; Male; Necrosis; Probability; Random Allocation; Rats; Rats, Zucker; Reference Values; Reperfusion Injury; Statistics, Nonparametric; Survival Rate; Tacrolimus; Time Factors

Ischemia reperfusion (I/R)-associated early graft pancreatitis is a major complication after pancreas transplantation. The influence of immunosuppressants on graft pancreatitis remains unclear. The aim of this study was to evaluate ciclosporin and tacrolimus in experimental pancreatic I/R.. Moderate pancreatitis was induced in rats by I/R injury. Animals were assigned to 4 groups: (1) control without I/R, (2) I/R without therapy, (3) I/R + ciclosporin, or (4) I/R + tacrolimus. After 24 hours, pancreatic damage was evaluated by amylase, endothelin 1, thromboxane A2, and histology. Additionally, microcirculation was evaluated 12 hours after reperfusion by intravital microscopy.. I/R significantly increased amylase compared with controls, with maximum levels after ciclosporin treatment. Histology showed comparable tissue injury in control and tacrolimus-treated animals. Ciclosporin-treated animals developed significantly (P < 0.05) more inflammation and necrosis compared with the other groups. Erythrocyte velocity evaluated by intravital microscopy was reduced in all animals after I/R. This was significantly pronounced after ciclosporin application. There was a significant increase of adherent leukocytes and platelets in ciclosporin-treated animals compared with both other groups.. Tacrolimus does not negatively influence I/R-induced pancreatitis, whereas ciclosporin aggravates pancreatic tissue damage after I/R. These effects should be evaluated in the clinical setting of pancreas transplantation.

Topics: Acute Disease; Animals; Blood Pressure; Cyclosporine; Disease Models, Animal; Heart Rate; Immunosuppressive Agents; Pancreas; Pancreas Transplantation; Pancreatitis; Rats; Reperfusion Injury; Tacrolimus

Ischemia-reperfusion injury affects posttransplant renal function, directly increases the probability of acute rejection, and is associated with chronic rejection and impaired long-term function. Animal studies suggest that ischemic preconditioning enhances resistance to ischemia and may be augmented by treating donors using immunosuppressant agents. This study sought to confirm the hypothesis that a combination of ischemic training and immunosuppression prior to renal harvest would maximize a transplanted kidney's resistance to ischemia-reperfusion injury.. Landrace pigs underwent either preharvest immunosuppression plus left kidney ischemic training (group 1, n=6) or ischemic training alone (group 2, n=6). Immunosuppression was composed of mycophenolate mofetil (20 mg/kg) and tacrolimus (0.1 mg/kg) administered intravenously 30 minutes before training. Training comprised 2 cycles of left renal pedicle occlusion for 5 minutes followed by release (reperfusion) for 10 minutes. Warm renal ischemia was then induced by clamping the left renal pedicle for 30 minutes, followed by heterotopic left kidney transplantation. Blood from the transplanted kidney renal vein was sampled directly at 0, 10, 20, 40, and 60 minutes posttransplantation for malondialdehyde (a reactive oxygen species marker), tumor necrosis factor-alpha (TNF-alpha), interleukins 6 and 8 (inflammatory cytokines), and erythrocyte-reduced glutathione (an antioxidant). Renal histology was graded on a 3-point scale.. Reperfusion levels of malondialdehyde, TNF-alpha, and interleukin 6 were significantly lower in group 1 at both 40 and 60 minutes. None of the animals in group 1 (0/6) that received preharvest immunosuppression showed severe interstitial inflammation, compared with 4 of 6 animals in group 2 that did (P<.03).. Preharvest immunosuppression with mycophenolate mofetil and tacrolimus significantly decreases immediate posttransplant reactive oxygen species and inflammatory cytokine production, enhances the protective effect of ischemic training, and should not only reduce ischemiareperfusion injury in transplanted kidneys but also should enhance immediate and long-term graft function while preventing acute rejection.

Topics: Animals; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Malondialdehyde; Mycophenolic Acid; Preoperative Care; Reperfusion Injury; Swine; Tacrolimus; Treatment Outcome

Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia.

Topics: Animals; Apoptosis; Astrocytes; Blotting, Western; Caspases; Cell Size; Cell Survival; Cells, Cultured; Cerebral Cortex; DNA Fragmentation; Enzyme Activation; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Glutamic Acid; In Situ Nick-End Labeling; Indoles; Infarction, Middle Cerebral Artery; Male; Membrane Potentials; Mitochondria; Neuroprotective Agents; Rats; Reperfusion Injury; Tacrolimus; Tetrazolium Salts; Thiazoles

Immunosuppressant FK506 is neuroprotective in experimental models of cerebral ischemia, but the molecular mechanisms underlying this neuroprotection remain unknown. We have demonstrated that FK506 inhibits the signaling pathways that regulate hypertrophic/proliferative responses in cultured astrocytes. Ischemia/reperfusion injury is associated with the proliferation and hypertrophy of astrocytes and with inflammatory responses. In the present work, we sought to determine whether FK506 neuroprotection after middle cerebral artery occlusion (MCAo) in rat is mediated via suppression of glia activation and changes in cytokine expression. Neurological deficits, infarct size, and astrocyte/microglial response were quantified in rats subjected to 90 min of MCAo. Changes in the mRNA expression of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) in ipsilateral and contralateral cortices were determined by reverse transcription-polymerase chain reaction (RT-PCR). FK506 administered at 1 mg/kg, 60 min after MCAo, produced a significant improvement in neurological function and reduction of infarct volume. In FK506-treated rats, a significant reduction of IL-1beta, IL-6, and TNF-alpha expression was observed 12 h after reperfusion. FK506 neuroprotection was associated with a significant downregulation of IL-1beta expression in astrocytes and microglia in the injured side. FK506 selectively decreased the levels of TNF-alpha, and IL-1beta mRNAs in astrocytes in vitro, with no effect on transforming growth factor-beta 1 (TGF-beta1) and IL-6 expression. Moreover, FK506 inhibits lipopolysaccharide (LPS)-induced activation and cytokine expression in microglia in vitro. Our findings suggest that astrocytes and microglia are targets for FK506, and that modulation of glial response and inflammation may be a mechanism of FK506-mediated neuroprotection in ischemia.

Topics: Animals; Animals, Newborn; Astrocytes; Brain; Cells, Cultured; Cerebral Infarction; Cytokines; Disease Models, Animal; Down-Regulation; Gliosis; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Interleukin-1; Interleukin-6; Ischemic Attack, Transient; Lipopolysaccharides; Male; Microglia; Neuroglia; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Tacrolimus; Tumor Necrosis Factor-alpha

Ischemia-reperfusion injury is an unavoidable problem for organ transplantation including small bowel transplantation, and causes a large intra-individual variation of tacrolimus (FK506) pharmacokinetics. Little information is available about the regulation of the intestinal P-glycoprotein expression during tissue regeneration. In the present study, we have examined the molecular and functional variations of ileum P-glycoprotein using rats after ischemia-reperfusion treatment. Morphological study revealed a rapid regeneration of the intestinal wall during 24 h after reperfusion. A reverse transcription-coupled competitive PCR and Western blot analysis revealed that the intestinal expression of P-glycoprotein recovered with time after reperfusion. At 24 h after reperfusion, the ileum P-glycoprotein level was transiently increased to two-fold, and the absorption rate of dihydro-[(3)H]FK506 from in situ ileum loop into portal vein was markedly low in comparison with the control. P-glycoprotein was detected in the crypt area as well as in villous cells at 6 h after reperfusion, and then localized to the apical surface at 24 h consistent with the cell proliferation and differentiation. However, the P-glycoprotein level returned to normal at 48 h. The intra-individual variation in the absorptive rate of tacrolimus was suggested to be regulated by the morphological status of the intestinal epithelium and enterocyte expression level of P-glycoprotein. Therefore, the monitoring of the enterocyte P-glycoprotein level would provide useful information for determining the dosage of tacrolimus immediately after small bowl transplantation.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Basement Membrane; Biological Transport; Ileum; Immunohistochemistry; Immunosuppressive Agents; Intestinal Absorption; Male; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Tacrolimus; Up-Regulation

We have previously shown the long-term influence of renal ischemia/reperfusion (I/R) injury and immunosuppression on fibrotic genes and apoptosis in a rat model. For the first time, we have now investigated the effects of I/R and immunosuppression on inflammation and caspase activation.. I/R injury was induced in the right kidney and the left was removed. Cyclosporin (CsA) (10 mg/kg), tacrolimus (0.2 mg/kg), rapamycin (1 mg/kg), or mycophenolate mofetil (MMF) (10 mg/kg) was then administered for 16 weeks. The effects of I/R and immunosuppressants on interstitial inflammation, interleukin (IL)-1beta expression, caspase-1 and caspase-3 activation, tubulointerstitial damage, and fibrosis were evaluated.. ED-1+ (a specific rat monocyte/macrophage marker) cells were mainly localized in the tubulointerstitium and periglomerular areas and increased in I/R group compared to controls (P < 0.01). This was further increased by CsA, but decreased by tacrolimus, rapamycin, or MMF (P < 0.05). The 17 kD active IL-1beta remained unchanged, but 35 kD IL-1beta precursor was decreased by rapamycin in comparison with I/R group (P < 0.05). The 45 kD or 20 kD caspase-1 was increased by I/R or CsA, respectively, and decreased by rapamycin (P < 0.05). The 24 kD caspase-3, which proved to be an active caspase-3 subunit, was increased in I/R and CsA groups and deceased by tacrolimus, rapamycin, or MMF (P < 0.05), but not 32 kD precursor or 17 kD active caspase-3. The activity data of caspase-1 and caspase-3 exhibited the same trend as Western blotting data. The staining of active caspase-3 was scattered in kidneys, mainly in tubular and interstitial areas, which was consistent with that of ED-1+ cells. There was a strong positive correlation between interstitial inflammation and 24 kD caspase-3 expression or caspase-3 activity (r = 0.814 or 0.484), all of which were also closely related with urinary protein (r = 0.537, 0.529, or 0.517), serum creatinine (r = 0.463, 0.573, or 0.539), tubulointerstitial damage (r = 0.794, 0.618, or 0.712) and fibrosis (r = 0.651, 0.567, or 0.469), all P < 0.01.. This study shows that the mechanisms of long-term I/R injury and immunosuppressants treatment include interstitial inflammation and caspase activation, most clearly demonstrated by the 24 kD active caspase-3.

Topics: Animals; Apoptosis; Caspase 1; Caspase 3; Caspases; Chronic Disease; Cyclosporine; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Fibrosis; Glomerulonephritis; Immunosuppressive Agents; Interleukin-1; Male; Mycophenolic Acid; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; Tacrolimus

In our experimental study, we aimed to test the effect of FK506, azathioprine and L-carnitine on protection of spinal cord injury due to ischemia-reperfusion.. Twenty-seven Sprague-Dawley male rats were randomly divided into five groups. They were subjected to spinal cord ischemia by clamping the abdominal aorta for 45 min. Thirty minutes before the aortic clamping, group I received 0.5 mg/kg FK506, group II received 100 mg/kg L-carnitine, group III received 4 mg/kg azathioprine, the fourth group was the control group and received only normal saline injection intravenously and the last group was the sham group. Neurological status was scored by using the Tarlov scoring system. Sections of the lumbar cord were harvested for histopathological grades (1-4), having regard to percentage of the apoptotic cells.. Hind-limb motor function had recovered normally 48 h after the operation in all rats which received FK506, azathioprine and L-carnitine prophylactically. In contrast, all rats in the control group had deteriorated to paraplegia by 48 h after the operation (P<0.05). Histopathologic sections in the involved spinal cord segment showed that a greater number of motor neuron cells were preserved and there were less apoptotic cells in the rats that received FK506, azathioprine and L-carnitine than those in control group.. These results suggest that prophylactic use of FK506, azathioprine and L-carnitine protects motor neuron cells from ischemic spinal cord injury.

Topics: Animals; Azathioprine; Carnitine; Drug Therapy, Combination; Immunohistochemistry; Immunosuppressive Agents; Male; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spinal Cord Ischemia; Tacrolimus

We previously demonstrated that calcineurin inhibitors given intravenously ameliorate experimental lung ischemia-reperfusion injury. This study evaluates whether these effects can be achieved when these agents are delivered endotracheally.. Left lungs of Long Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received tacrolimus endotracheally at doses of 0.2, 0.1, or 0.025 mg/kg 60 minutes before ischemia. Injury was quantitated in terms of vascular permeability. Additional animals treated at a dose of 0.1 mg/kg were assessed for lung tissue myeloperoxidase content and bronchoalveolar lavage leukocyte content. Bronchoalveolar lavage fluid was assessed for cytokine and chemokine content by enzyme-linked immunosorbent assay. Tissue samples were processed for nuclear factor-kappaB activation, and blood levels of tacrolimus were measured in treated animals.. Left lung vascular permeability was reduced in treated animals in a dose-dependent fashion compared with controls. The protective effects correlated with a 47% (0.50% +/- 0.06% vs 0.27% +/- 0.08%, respectively) reduction in tissue myeloperoxidase content (P <.004) and marked reductions in bronchoalveolar lavage leukocyte accumulation. This protection was also associated with decreased nuclear factor-kappaB activation and diminished expression of proinflammatory mediators. Blood tacrolimus levels in treated animals at 4 hours of reperfusion were undetectable.. Tacrolimus administered endotracheally is protective against lung ischemia-reperfusion injury in our model. This protection is associated with a decrease in nuclear factor-kappaB activation. This route of tacrolimus administration broadens its potential clinical use and decreases concerns about systemic and renal toxicity. It may be a useful therapy in lung donors to protect against lung ischemia-reperfusion injury.

Topics: Animals; Bronchoalveolar Lavage Fluid; Calcineurin; Capillary Permeability; Cell Count; Chemokines, CXC; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Immunosuppressive Agents; Intercellular Signaling Peptides and Proteins; Lung; Male; Models, Cardiovascular; NF-kappa B; Peroxidase; Rats; Rats, Long-Evans; Reperfusion Injury; Tacrolimus; Treatment Outcome

Topics: Animals; Apoptosis; Calcineurin Inhibitors; Cyclosporine; Male; Neuroprotective Agents; Rabbits; Reperfusion Injury; Spinal Cord Ischemia; Tacrolimus

The study investigated the possibility of pharmacologically modulating hepatic allograft function from non-heart-beating donors (NHBDs) using male Lewis rats. The donors were divided into 4 groups: Group 1 in which the vehicle was administered, Group 2 in which FK506 (tacrolimus; a powerful immunosuppressive agent) was administered, Group 3 in which OKY046 (a specific thromboxane synthetase inhibitor) was administered and Group 4 in which FK506 and OKY046 were administered. The recipients received orthotopic liver transplantation. The survival rates differed significantly between the recipients that had received liver transplantation from Groups 1 and 4. The serum liver enzyme and inflammatory cytokine concentrations of the recipients which had received liver transplantation from Groups 2, 3 and 4 were significantly lower than those of the recipients that had received liver transplantation from Group 1. Although there was no significant difference, all parameters were better in the recipients that had received transplantation from Group 4 than those that had received transplantation from Groups 2 and 3. The action mechanisms of FK506 and OKY046 are completely different. Therefore, concomitant use of FK506 and OKY046 might have additive effects on liver transplantation from NHBDs. In conclusion, we demonstrated that pretreatment of NHBDs using FK506 and OKY046 ameliorated graft viability.

Topics: Animals; Enzyme Inhibitors; Graft Survival; Immunosuppressive Agents; Liver; Liver Transplantation; Male; Methacrylates; Rats; Rats, Inbred Lew; Reperfusion Injury; Tacrolimus; Thromboxane-A Synthase; Tissue Donors; Transplantation, Homologous

Topics: Animals; Azathioprine; Carnitine; Neuroprotective Agents; Rats; Reperfusion Injury; Spinal Cord Ischemia; Tacrolimus

FK506 (tacrolimus), an immunosuppressant, reportedly reduces ischemic brain injury following transient middle cerebral artery occlusion (MCAO) in rats. The authors previously reported that the therapeutic window of FK506 in this model is more than 1 h, but less than 2 h. The aim of the present study is to determine whether mild hypothermia (35 degrees C) enhances the neuroprotective effects of FK506 and expands its therapeutic window. Sprague-Dawley rats were subjected to 2 h MCAO followed by 24 h reperfusion. Animals were randomly divided into four groups: (I) vehicle-treated normothermic group; (II) FK506-treated normothermic group; (III) vehicle-treated hypothermic group; (IV) FK506-treated hypothermic group. Animals received a single injection of FK506 (0.3 mg/kg) or vehicle intravenously at 2 h after ischemic induction. During ischemia, temporal muscle and rectal temperatures were maintained at 37 degrees C in the normothermic animals and at 35 degrees C in the hypothermic animals. Infarct volumes and neurological performance were evaluated at 24 h after reperfusion. The combination of FK506 and mild hypothermia significantly reduced infarct volume (cortex, -61%; striatum, -31%) and edema volume (cortex, -57%; striatum, -41%), while mild hypothermia or FK506 alone failed to improve ischemic brain damage. Furthermore, this combination also provided for the best functional outcome. These results demonstrate that the combination of FK506 and mild hypothermia significantly reduces ischemic brain damage following transient MCAO in rats, and expands the therapeutic window for FK506. This therapy may be a new approach for treatment of acute stroke.

Topics: Animals; Body Temperature; Brain Edema; Cerebral Infarction; Cerebrovascular Circulation; Hypothermia, Induced; Immunosuppressive Agents; Ischemic Attack, Transient; Laser-Doppler Flowmetry; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Ischemia/reperfusion (I/R) carries significant injury to endothelial cells in transplanted organs and is an important factor in chronic rejection. Immunosuppressive drugs, notably cyclosporin A (CyA) and FK506, can potentially augment this injury. Here, our goal was to determine the combined effects of I/R and CyA or FK506 on endothelial cells. Transformed mouse endothelial cells (SVEC 4-10) were subjected to ischemia or I/R for 2-24 hours by incubating cells in 100 per cent N2 (ischemia) followed by 5 per cent CO2 and 95 per cent O2 (reperfusion) for 24 hours. In separate experiments, CyA or FK506 was added to cells subjected to ischemia or I/R. Nonviable cells were determined by Trypan blue exclusion assay. All experiments (done in triplicate) were analyzed by Student's t test. Increasing ischemia times resulted in a greater number of nonviable cells (2% nonviable cells at 0 hours and 57% at 24 hours of I/R). Addition of CyA significantly increased the number of nonviable cells when compared with the control (I/R only) group (P = 0.014). Interestingly, FK506 did not increase the percentage of nonviable cells compared with the control group (P = 0.2). Unlike FK506, CyA augments I/R injury to endothelial cells in vitro. These findings could be relevant in chronic rejection and transplantation.

Topics: Analysis of Variance; Animals; Apoptosis; Calcineurin Inhibitors; Cell Count; Cell Line, Transformed; Cell Survival; Cells, Cultured; Coloring Agents; Cyclosporine; Endothelial Cells; Graft Rejection; Immunosuppressive Agents; Ischemia; Mice; Necrosis; Reperfusion Injury; Tacrolimus; Time Factors; Trypan Blue

This study investigated the effect of gangliosides (Gang) on small bowel microcirculation and animal survival after normothermic intestinal ischemia-reperfusion injury. Five adult male EPM-1 Wistar rats in each of three groups received FK506 (0.2 mg/kg), Gang (3 mg/kg), or vehicle (at same volume) either 24 or 12 hours prior to the experiment. The animals were anesthetized intramuscularly with ketamine (60 mg/kg) and xylazine (10 mg/kg) and hydrated with 80 mL/kg of prewarmed saline solution delivered subcutaneously before the ischemic insult and 40 mL/kg at 1 hour after reperfusion. Under anesthesia, they underwent a laparotomy with clamping of the superior mesenteric artery (SMA) at its origin for 75 minutes. Microcirculation was evaluated with a laser Doppler flowmeter, 5 minutes before ischemia (baseline) and reperfusion (ischemia), and 20, 40, and 60 minutes after reperfusion. Animal survival was observed up to 24 hours. Small bowel flow measured before ischemia was considered to be the baseline level (100%). After SMA occlusion a significant reduction in microcirculatory tissue perfusion to about 8% was observed in all groups. At 20, 40, and 60 minutes of reperfusion treatment with Gang (77%, 81%, and 100%) or FK506 (70%, 85%, and 98%) promoted better recovery of the intestinal microcirculation when compared to the control group (45%, 72%, and 75%). Concerning animal survival there was no difference between groups (just one animal from each group, Gang and FK506, survived up to 24 hours). Based on our data we conclude that Gang and FK506 improve intestinal microcirculation in ischemia-reperfusion injury but do not change animal survival after severe ischemia.

Topics: Animals; Flow Cytometry; Gangliosides; Immunosuppressive Agents; Intestines; Male; Microcirculation; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus

Hindlimb ischemia and reperfusion leads to lung injury in various animal models. We investigated the effectiveness of FK506, an immunosuppressive agent, which also modulates neutrophilic infiltration, in preventing lung injury after hindlimb ischemia and reperfusion in a rat model.. Twenty-seven male Sprague-Dawley rats were randomized to received FK506 at doses of 0.3 mg/kg, 0.5 mg/kg, or 1 mg/kg body weight per day, or normal saline injections, as pretreatment, and there was also a sham group. On the 4th day, the animals were subjected to 2 h of ischemia induced by a tourniquet, followed by reperfusion of the extremities for 2 h. Lung tissue assays were performed for the lipid peroxidation product malondialdehyde (MDA) and total glutathione (GSH). Lung tissues were also examined histopathologically under light and electron microscopy.. The MDA levels in the study groups were significantly lower than those in the control group ( P < 0.05), but the total GSH levels did not differ significantly among the groups. Histopathologically, there were no significant differences among the groups given different doses of FK506, but there was a significant difference between the control group and all the treatment groups.. FK506 ameliorates the lung injury associated with ischemia and reperfusion of the lower limbs, and might have an inhibitory effect on the neutrophils that cause remote organ damage.

Topics: Animals; Glutathione; Hindlimb; Immunosuppressive Agents; Lipid Peroxidation; Lung; Male; Malondialdehyde; Microscopy, Electron, Transmission; Neutrophils; Premedication; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

The aim of this study was to determine whether the minimum necessary volume of a moderate fatty liver graft was similar to the normal liver volume and to elucidate means for improving the function of the transplanted fatty liver if it were inferior in volume to a normal liver under conditions of permissible cold preservation. Nine-week-old male Wistar rats were used. Normal rat chow was fed to the normal liver group, and fat-enriched rat chow was fed to the fatty liver group for 4 weeks to induce a moderately fatty liver. Liver transplantation with various volumes of reduced-size grafts, including whole liver graft (100%LT), 70% volume graft (70%LT), and 30% volume graft (30%LT), was performed with both groups of rats as donors. All procedures were performed under the conditions of 2-hour cold preservation. All rats with an implanted normal liver were surviving at 7 days after the operation regardless of the graft volume (100%LT, 5 of 5; 70%LT, 5 of 5; 30%LT, 5/5). In contrast, the survival rates decreased according to the graft volume in rats implanted with fatty livers (100%LT, 8 of 8; 70%LT, 5 of 8; 30%LT, 2/8). To improve the survival of 30%LT with fatty liver, we employed two potent inhibitors of ischemia-reperfusion injury: FK506 and prostaglandin E1. Though FK506 had no advantageous effect, prostaglandin E1 significantly improved the survival rate and diminished serum levels of alanine aminotransferase and hyaluronic acid. In conclusion, the volume of graft necessary for successful transplantation is larger in fatty livers than in normal livers in permissible cold preservation. Also, prostaglandin E1 protects grafts against ischemia-reperfusion injury and improves the functioning of a transplanted fatty liver.

Topics: Alanine Transaminase; Alprostadil; Animals; Fatty Liver; Hyaluronic Acid; Immunosuppressive Agents; Liver; Liver Transplantation; Male; Models, Animal; Organ Preservation; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus

The signaling pathways of mitogen-activated protein kinases (MAPKs) are important molecular components responsible for ischemia/reperfusion (I/R) injury in the kidneys. Preconditioning with cyclosporine A (CsA) or FK506 reduces subsequent I/R injury. We studied the effect of preconditioning with CsA or FK506 on MAPK expression in ischemic rat kidneys.. Two separate studies were performed using Sprague-Dawley rats. First, MAPK (extracellular signal-regulated kinase [ERK], jun N-terminal kinase [JNK], p38) expressions were observed at 0, 10, 20, 30, 60, 120, and 1,440 min after I/R injury. Second, the effects of preconditioning with CsA or FK506 on MAPK expressions were observed in rat kidneys with I/R injury. I/R injury was induced by clamping both renal arteries for 45 min. Rats were pretreated with intravenous (IV) CsA (3 mg/kg) or IV FK506 (0.3 mg/kg) 6 hr before I/R injury and killed 30 min later. Expression of MAPK was measured using immunoblot and immunohistochemistry.. MAPK (ERK, JNK, p38) expressions were significantly increased in kidneys with I/R injury compared with sham-operated controls, and immunohistochemistry revealed increased MAPK immunoreactivity in renal tubules of the outer medulla. Kidneys preconditioned with low-dose CsA or FK506 showed significantly increased ERK expression compared with kidneys with I/R injury alone (CsA, 9.5- vs. 4.5-fold; FK506 10.4- vs. 4.5-fold: P<0.05) but showed decreased JNK (CsA, 3.8- vs. 5.3-fold; FK506, 3.4- vs. 5.3-fold: P<0.05) and p38 expression (CsA, 2.5- vs. 3.7-fold; FK506, 2.1- vs. 3.7-fold: P<0.05).. Preconditioning with CsA or FK506 differentially regulates the expression of MAPK in rat kidneys with I/R injury, and this may explain the remarkable protective effects of these agents.

Topics: Animals; Cyclosporine; Immunosuppressive Agents; Ischemia; Ischemic Preconditioning; JNK Mitogen-Activated Protein Kinases; Kidney; Male; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Grafts from so-called "marginal donors" are increasingly used for organ transplantation. The combination of reduced organ quality and additional inflammatory damages may be particularly detrimental in these grafts. In a previous study, we showed the beneficial effects on long-term graft outcome of "suboptimal" grafts by the induction of heme oxygenase-1. Here we tested the impact of short-term donor treatment with established immunosuppressants.. Twelve-month-old Fischer 344 donor rats either were treated with prednisolone, mycophenolate mofetil, RAD, or FK506 24 hr and 1 hr before organ harvesting or remained untreated. Renal allografts were perfused with University of Wisconsin solution and kept at 4 degrees C for an ischemic period of 2 hr. Morphologic, immunohistologic, and real time reverse transcriptase-polymerase chain reaction analyses for relevant markers were performed at serial intervals and at the end of the observation period (6 months).. All animals survived the observation period, although the ischemic time resulted in accelerated chronic graft dysfunction. Grafts from donors treated with prednisolone or FK506 demonstrated significantly improved graft function and structure by 6 months. Mononuclear infiltrates were significantly reduced by the end of the observation period, whereas intragraft mRNA levels of tumor necrosis factor-alpha and interleukin-10 were significantly altered during the early period after transplantation. Minor improvements in graft function and histologic alterations of suboptimal grafts were observed after pretreatment with mycophenolate mofetil and RAD.. Donor treatment with approved immunosuppressants, in particular prednisolone or FK506, represents a novel therapeutic strategy of clinical relevance, most importantly when using grafts from marginal donors.

Topics: Age Factors; Animals; Antigens, Surface; Cytokines; Gene Expression; Graft Survival; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Immunosuppressive Agents; Kidney Transplantation; Male; Mycophenolic Acid; Prednisolone; Protoporphyrins; Rats; Rats, Inbred F344; Reperfusion Injury; Tacrolimus; Tissue Donors; Transplantation, Homologous

Microcirculatory disturbances following small intestinal ischaemia-reperfusion (I/R) injury lead to tissue damage that may affect short- and long-term outcome after transplantation. The immunosuppressive drug Tacrolimus (FK506) attenuates I/R injury in a number of organs, raising the possibility that it might be able to control both I/R injury and rejection after small bowel transplantation. However, its effects on intestinal I/R injury have not been evaluated.. PVG rats were subjected to 30 min intestinal ischaemia. Animals received Tacrolimus (1 mg/kg i.p.) 4 and 1 h prior to ischaemia. The mucosa was visualised in an exteriorised ileal segment using in vivo microscopy. FITC-BSA or Acridine orange was used to quantitate macromolecular leak (MML) and leucocyte adhesion respectively every 15 min for 2 h during reperfusion. Heart rate and mean blood pressure (mBP) were monitored throughout the experiment.. Ten of 12 untreated animals subjected to intestinal I/R injury failed to survive the 2-hour reperfusion period. MML and leucocyte adhesion were increased in untreated animals (p < 0.001) and blood flow stasis eventually ensued. Similar results were obtained for Tacrolimus pre-treated I/R animals, with 10 of 12 animals again failing to survive reperfusion.. Despite previous evidence that Tacrolimus reduces I/R injury in other organs, it did not improve survival rates or prevent villus microcirculatory disturbances following intestinal I/R injury. The severity of microcirculatory damage suffered by the small intestine highlights the importance of alternative therapies to combat I/R in this organ.

Topics: Animals; Capillary Permeability; Cell Adhesion; Immunosuppressive Agents; Intestinal Mucosa; Male; Microcirculation; Microscopy; Rats; Reperfusion Injury; Splanchnic Circulation; Tacrolimus

The aim of this study was to demonstrate that tacrolimus (FK506) has a hepatoprotective effect by reducing ischemia-reperfusion-induced apoptosis and necrosis, both of which lead to post-surgical liver dysfunction. An ischemia-reperfusion model and primary cultured rat hepatocytes subjected to hypoxic and reoxygenation phases, mimicking the surgical process, were used. c-Jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK1/SAPK1) activation leads to caspase 3 activation, a trigger of apoptosis. The activation status of JNK1/SAPK1 was evaluated by immunoprecipitation or Western-blotting experiments. Apoptosis was assessed by measuring caspase activation and by TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling) reaction. Necrosis was assessed histologically. Tacrolimus improved the survival rate of rats subjected to ischemia-reperfusion. After FK506 pretreatment, the liver necrosis rate was reduced, and ischemia-reperfusion-induced JNK1/SAPK1 activation and apoptosis were significantly decreased. In hypoxia-reoxygenation-subjected hepatocytes, tacrolimus reduced JNK1/SAPK1 and caspase 3 activation. In the liver, tacrolimus prevented ischemia-reperfusion-induced apoptosis and necrosis.

Topics: Animals; Apoptosis; Blotting, Western; Caspases; Cells, Cultured; Hepatocytes; Immunosuppressive Agents; In Situ Nick-End Labeling; Ischemia; JNK Mitogen-Activated Protein Kinases; Liver; Male; Mitogen-Activated Protein Kinases; Necrosis; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus

Epidermal growth factor (EGF) plays an important role in tubular regeneration in kidneys with ischemia/reperfusion (I/R) injury. This study was undertaken to evaluate the influence of cyclosporine A (CsA) or FK506 on mature EGF expression and tubular regeneration in rat kidneys with I/R injury.. Two separate studies were performed. First, the expression of EGF and tubular regeneration was observed in rat kidneys with I/R injury on days 1, 2, 3, 5, and 7. Second, the dose-dependent response of EGF expression and tubular regeneration to CsA (5, 10, and 20 mg/kg) or FK506 (0.25, 0.5, and 1.0 mg/kg) was observed in rat kidneys with I/R injury. I/R injury was induced by clamping both renal arteries for 45 min, and CsA or FK506 was injected just after release of vascular clamps. Rats were sacrificed on day 1 for evaluation of EGF expression, and on day 2 for evaluation of BudU-positive cells. Renal function, tubular injury score, EGF expression assessed by immunoblotting, levels of CsA and FK506 in whole blood, and immunostaining for BrdU was studied.. EGF expression was maximal on day 1 (cortex, 29-fold; medulla, 31-fold compared with sham-operated controls), and renal tubular regeneration measured with the number of BrdU-positive cells was maximal on days 2 and 3 in kidney with I/R injury, and thereafter the level of EGF and the number of BrdU-positive cells decreased progressively. CsA or FK506 treatment to ischemic rat kidneys reduced the expression of EGF and the number of BrdU-positive cells in a dose-dependent manner.. CsA or FK506 treatment delays recovery from acute tubular necrosis, and this may be associated with decreased EGF expression by CsA or FK506.

Topics: Animals; Antimetabolites; Bromodeoxyuridine; Cyclosporine; Epidermal Growth Factor; Immunosuppressive Agents; Kidney Tubules; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Elevated levels of free fatty acids (FFA) have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the effects of immunosuppressants agents, calcineurin inhibitors and blockade of endoplasmic reticulum (ER) calcium channels on free fatty acid formation and efflux in the ischemic/reperfused (I/R) rat brain. Changes in the extracellular levels of arachidonic, docosahexaenoic, linoleic, myristic, oleic and palmitic acids in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical cup technique. A 20-min period of ischemia elicited large increases in the efflux of all six FFAs, which were sustained during the 40 min of reperfusion. Cyclosporin A (CsA) and trifluoperazine, which reportedly inhibit the I/R elicited opening of a mitochondrial permeability transition (MPT) pore, were very effective in suppressing ischemia/reperfusion evoked release of all six FFAs. FK506, an immunosuppressant which does not directly affect the MPT, but is a calcineurin inhibitor, also suppressed the I/R-evoked efflux of FFAs, but less effectively than CsA. Rapamycin, a derivative of FK506 which does not inhibit calcineurin, did not suppress I/R-evoked FFA efflux. Gossypol, a structurally unrelated inhibitor of calcineurin, was also effective, significantly reducing the efflux of docosahexaenoic, arachidonic and oleic acids. As previous experiments had implicated elevated Ca(2+) levels in the activation of phospholipases with FFA formation, agents affecting endoplasmic reticulum stores were also evaluated. Dantrolene, which blocks the ryanodine receptor (RyR) channel of the ER, significantly inhibited I/R-evoked release of docosahexaenoic, arachidonic, linoleic and oleic acids. Ryanodine, which can either accentuate or block Ca(2+) release, significantly enhanced ischemia/reperfusion-elicited efflux of linoleic acid, with non-significant increases in the efflux of myristic, arachidonic, palmitic and oleic acids. Xestospongin C, an inhibitor of the inositol triphosphate (IP(3)R) channel, failed to affect I/R-evoked FFA efflux. Thapsigargin, an inhibitor of the Ca(2+)-ATPase ER uptake pump, elicited significant elevations in the efflux of myristic, arachidonic and linoleic acids, in the absence of ischemia. Collectively, the data suggest an involvement of both ER and mitochondrial Ca(2+) stores in the chain of events which lead to PLA(2) activation and

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Calcium Channels; Cerebral Cortex; Cyclosporine; Dantrolene; Endoplasmic Reticulum; Enzyme Inhibitors; Fatty Acids, Nonesterified; Gossypol; Immunosuppressive Agents; Macrocyclic Compounds; Male; Oxazoles; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ryanodine; Sirolimus; Tacrolimus; Thapsigargin; Trifluoperazine

Topics: Acute Disease; Animals; Biopsy; Creatinine; Heart Arrest; Immunosuppressive Agents; Kidney; Kidney Diseases; Kidney Function Tests; Liver; Liver Function Tests; Liver Transplantation; Reperfusion Injury; Swine; Tacrolimus; Transplantation, Homologous

Calcineurin inhibitors reduce experimental reperfusion injury in the liver, brain, heart, kidney, and small bowel. These studies were undertaken to determine whether these agents are similarly protective against lung ischemia-reperfusion injury.. Left lungs of male rats were rendered ischemic for 90 minutes and reperfused for as long as 4 hours. Treated animals received cyclosporine A (INN: ciclosporin; 1 or 5 mg/kg) or tacrolimus (0.2 mg/kg) 6 hours before ischemia, at reperfusion, or 2 hours after reperfusion. Injury was quantitated in terms of tissue polymorphonuclear leukocyte accumulation (myeloperoxidase content), vascular permeability (iodine 125-labeled bovine serum albumin extravasation), and bronchoalveolar lavage leukocyte content. Separate tissue samples were processed for nuclear protein and cytokine messenger RNA.. Treatment with cyclosporine (5 mg/kg) or tacrolimus (0.2 mg/kg) 6 hours before reperfusion reduced lung vascular permeability by 54% and 56% relative to control animals (P <.03). The protective effects of cyclosporine and tacrolimus treatment before reperfusion correlated with 42% and 43% reductions in tissue polymorphonuclear leukocyte (myeloperoxidase) content (P <.008) and marked reductions in bronchoalveolar lavage leukocyte accumulation (P <.01). Administration of cyclosporine or tacrolimus at the time of reperfusion or 2 hours into the reperfusion period offered little or no protection. Animals treated before reperfusion also demonstrated marked reductions in nuclear factor kappaB activation and expression of proinflammatory cytokine messenger RNA.. Cyclosporine and tacrolimus treatment before reperfusion was protective against lung ischemia-reperfusion injury in rats. The mechanism of these protective effects may involve the inhibition of nuclear factor kappaB, a central transcription factor mediating inflammatory injury. The decreased expression of cytokine messenger RNA indicates that both cyclosporine and tacrolimus may exert their protective effects at the pretranscriptional level.

Topics: Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cyclosporine; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Immunosuppressive Agents; Leukocytes; Lung; Male; NF-kappa B; Peroxidase; Preoperative Care; Rats; Rats, Long-Evans; Reperfusion Injury; RNA, Messenger; Tacrolimus; Treatment Outcome

The immunosuppressive drug FK506 (tacrolimus) has been reported to be a powerful neuroprotective agent in the focal ischemia of animals. However, no report has been published concerning neuroprotective effect of this compound on the morphology in superacute stage. The separate analysis between early and delayed effects of FK506 on the morphology may be helpful in the study of the compound's mechanism of action which is still unknown. The goal of this study was to determine early and delayed effects of pharmacological treatment with FK506 in permanent MCA occlusion using magnetic resonance imaging (MRI). Nineteen rats were subjected to permanent MCA occlusion, and given either intravenous injection of placebo or 1 mg/kg FK506 immediately after occlusion. DWI and T(2)-weighted MRI were performed 3 and 24 h after MCA occlusion, and postmortem histological analysis was also performed. FK506 drastically reduced the ischemic damage in 3-h apparent diffusion coefficient (ADC) map. This is the first report to demonstrate the neuroprotective effects of FK506 on focal cerebral ischemia in superacute stage. In addition, postmortem ischemic damage tended to be smaller than ischemic area indicated by 3-h ADC map in the FK506 group, whereas there was an excellent equality between them in the placebo group, suggesting the possible effect of FK506 on the later ischemic period. Our findings provide direct evidence for the neuroprotective effect of FK506 on ischemic cell damage in both early stage and possibly later stage.

Topics: Animals; Brain; Diffusion; Image Enhancement; Image Processing, Computer-Assisted; Infarction, Middle Cerebral Artery; Injections, Intravenous; Magnetic Resonance Imaging; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Tacrolimus (FK506), an immunosuppressant currently used in clinic, is known to have neuroprotective properties. However, effects in focal ischemia are shown only in a endothelin induced middle cerebral artery (MCA) occlusion model or with filament technique at a relatively high dose. We have previously shown that FK506 had significant protective effects at a low dose of 0.3 mg kg(-1) when administered immediately after ischemia. In this study, we explored the therapeutic time window of FK506 at this low dose, in a transient focal ischemia model using filament technique. Male Sprague-Dawley rats were subjected to 2 h MCA occlusion and subsequent reperfusion. They received FK506 or vehicle (0.3 mg kg(-1)) i.v. at 30, 60 or 120 min after induction of ischemia, and were decapitated 24 h after ischemia. FK506 injected at 30 and 60 min significantly reduced cortical infarction volume (FK506 vs. vehicle; 30 min: 95 +/- 33 mm3 vs. 170 +/- 62 mm3, p < 0.05; 60 min: 93 +/- 45 mm3, vs. 168 +/- 35 mm3, p < 0.05, respectively). FK506 was ineffective when given at 120 min after ischemia. FK506 had no effect on edema formation, nor on the infarct volume in striatum. The therapeutic time window for this low dose of FK506 given i.v. is between 60 and 120 min in this model.

Topics: Animals; Blood Glucose; Brain; Brain Edema; Brain Ischemia; Cardiovascular Physiological Phenomena; Cerebral Infarction; Drug Administration Schedule; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus; Treatment Outcome

Ischemia/reperfusion (I/R) injury in the early posttransplant period is closely associated with delayed recovery of graft function, increased acute rejection, and late allograft dysfunction. Pharmacological preconditioning with low-dose cyclosporine (CsA) or FK506 was performed to induce ischemic tolerance in rat kidney with I/R injury.. Low-dose CsA (3 mg/kg, administered i.v.) or FK506 (0.3 mg/kg i.v.) were used to induce ischemic tolerance in Sprague-Dawley rats, and the induction of heat shock protein (hsp) 70 by CsA or FK506 was evaluated overtime. Rats were pretreated with CsA or FK506 6 hr before I/R injury when hsp70 was maximally expressed, and were killed 24 hr later. The effect of pharmacological preconditioning on subsequent I/R injury was evaluated in terms of renal function, histopathology score, assays for apoptosis (DNA fragmentation analysis, TUNEL staining, expressions of pro-apoptotic genes, and caspase activity), and the expression of inflammatory cytokine genes (interleukin-1 and tumor necrosis factor-alpha).. Preconditioning with low-dose CsA or FK506 significantly improved renal function and renal histology, compared to rats with I/R injury. Apoptotic cell death (typical DNA laddering and increased TUNEL-positive cells) in rat kidneys with I/R injury, was decreased by pretreatment with low-dose CsA or FK506. Increased expression of pro-apoptotic genes (Fas, Fas-ligand, caspase 1 and 3) and activated caspases in ischemic rat kidneys were decreased after CsA or FK506 pretreatment.. Pretreatment with low-dose CsA or FK506 prevents subsequent I/R injury, and this effect may be related to the induction of hsp70. Pretreatment of renal donors with low-dose CsA or FK506 may result in an improvement in immediate posttransplant function.

Topics: Animals; Apoptosis; Blood Urea Nitrogen; Caspases; Creatinine; Cyclosporine; Dose-Response Relationship, Drug; Gene Expression; HSP70 Heat-Shock Proteins; Immunosuppressive Agents; Interleukin-1; Ischemia; Kidney Transplantation; Male; Rats; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Tacrolimus; Transplantation Conditioning; Tumor Necrosis Factor-alpha

FK506 is an immunosuppressive agent that has been reported to have neuroprotective effects in several kinds of rodent models of stroke. The purpose of this study was to evaluate the neuroprotective effects of FK506 in a monkey model of stroke.. Cynomolgus monkeys underwent 3 h of occlusion followed by 5 h of reperfusion of the right middle cerebral artery (MCA) through a transorbital approach. A single bolus dose of FK506 (0.1 mg/kg) was injected intravenously 5 or 175 min after MCA occlusion. Eight hours after ischemia, a neuropathologic study was performed and the volume of ischemic damage was determined. To measure local cerebral blood flow (CBF), the cerebral metabolic rate of oxygen (CMRO(2)), and the oxygen extraction fraction during the experiments, PET scans were obtained using a steady-state (15)O continuous-inhalation method. Four consecutive PET scans (before and 2 h after ischemia and immediately and 3 h after reperfusion) were obtained on each monkey.. Treatment with FK506 (0.1 mg/kg) 5 or 175 min after ischemia significantly reduced cortical damage 8 h after ischemia by 82% (P < 0.05) and 73% (P < 0.05), respectively. In PET studies, FK506 did not affect CBF or physiologic parameters in any treatments. In the FK506-treated group, a volume of >40% CMRO(2) reduction 3 h after reperfusion decreased significantly (P < 0.05).. This study showed that FK506 showed a powerful neuroprotective effect in a nonhuman primate model of stroke. The therapeutic time window of FK506 was at least 3 h after onset. PET studies detected neuroprotective effects only in areas with >40% CMRO(2) reduction 3 h after reperfusion.

Topics: Animals; Brain; Brain Ischemia; Macaca fascicularis; Male; Neuroprotective Agents; Reperfusion Injury; Tacrolimus; Tomography, Emission-Computed

Effects of the calcineurin inhibitor FK506, the platelet-activating factor (PAF) antagonist, and free radical scavenger Ginkgo biloba extract, EGb 761, and their combination on reperfusion-induced ventricular fibrillation (VF), ventricular tachycardia (VT), and recovery of cardiac function were studied after 30 min of global ischemia followed by 2 h of reperfusion in isolated rat hearts. In the first series of studies, rats received a daily (oral) dose of 0, 1, 5, 10, 20, or 40 mg/kg/day FK506 for 10 days. FK506 dose-dependently reduced the incidence of reperfusion-induced total (irreversible plus reversible) VF from a value of 92% for untreated animals to 92% (NS), 83% (NS), 67% (NS), 33% (p<0.05), and 25% (p<0.05), for doses of 1-40 mg/kg/day, respectively, with effects on incidence of VT showing the same pattern. FK506, between 20 and 40 mg/kg/day, also resulted in significant recovery of postischemic cardiac function. In the second series of studies, rats were treated with EGb 761 alone or in combination with FK506. Whereas no significant reduction in arrhythmias or improvement in cardiac function resulted from a single intervention of EGb 761 at 25 mg/kg/day, combined treatment of rats with 25 mg/kg/day of EGb 761 and 1 or 5 mg/kg/day of FK506 resulted in a reduction in total and irreversible VF of 92% and 92% to 42% (p<0.05) and 33% (p<0.05), 25% (p<0.05) and 8% (p<0.05), respectively, versus untreated control animals, paralleled by similar effects on the incidence of VT and accompanied by significant improvements in postischemic cardiac function. Our results demonstrate a novel cardioprotective characteristic of FK506 and suggest that combination therapy by using FK506 plus EGb 761 synergistically improves postischemic cardiac function, while reducing the incidence of reperfusion-induced VF and VT, which may expand the clinical utility of FK506 and allow therapy with FK506 at lower doses than are currently useful.

Topics: Animals; Calcineurin Inhibitors; Dose-Response Relationship, Drug; Electrocardiography; Flavonoids; Free Radical Scavengers; Ginkgo biloba; Hemodynamics; In Vitro Techniques; Male; Myocardial Reperfusion Injury; Plant Extracts; Plants, Medicinal; Platelet Activating Factor; Platelet Membrane Glycoproteins; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Reperfusion Injury; Tacrolimus

Cyclosporin is associated with significant chronic nephrotoxicity, manifest in the long term mainly as renal fibrosis. There have been claims that tacrolimus is a less fibrotic drug than cyclosporin, and this study was designed to determine the effect of the two drugs on the expression of fibrosis-associated genes.. Male Wistar rats underwent clamping of the right renal pedicle for 45 min together with left nephrectomy; this model has previously been shown to be associated with upregulation of fibrosis-associated genes. Experimental groups (six animals per group) received cyclosporin A 10 mg/kg daily, tacrolimus 0.2 mg/kg daily or no treatment. Animals were killed at 16 weeks, and the renal cortical expression of fibrosis-associated genes was studied by means of quantitative reverse transcriptase-polymerase chain reaction.. Tacrolimus-treated animals developed significantly less proteinuria and had lower serum creatinine levels than those receiving cyclosporin. Tacrolimus administration also significantly reduced the expression of transforming growth factor beta and tissue inhibitor of metalloproteinases 1, both the products of genes associated with fibrosis. Although cyclosporin treatment reduced levels of the matrix-degrading enzymes, matrix metalloproteinase (MMP) 2 and MMP-9, this was not statistically significant.. Tacrolimus has less nephrotoxicity than cyclosporin in this model. It also appears to have less fibrogenic potential, and this may have implications for the choice of long-term immunosuppressant in renal transplantation.

Topics: Animals; Creatinine; Cyclosporine; Fibrosis; Gene Expression; Immunosuppressive Agents; Kidney Diseases; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Proteinuria; Rats; Rats, Wistar; Renal Artery; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Tacrolimus; Transforming Growth Factor beta

Endothelin-1 (ET-1), a novel vasoconstrictor, possibly plays a role in the mediation of ischemia/reperfusion (I/R) injury. Tacrolimus (FK506) and cyclosporin A (CsA) were reported to maintain tissue microcirculation of the liver subjected to I/R. This study investigated the effects of these immunosuppressants on intestinal I/R in terms of intestinal tissue microcirculation associated with ET-1.. Male S-D rats were pretreated twice with FK506 (0.2 mg/kg), CsA (10 mg/kg) or only saline solution (0.5 mL). The tissue microcirculation in the control was reduced after I/R (29% +/- 10%) accompanied by hypotension, increased tissue ET-1 expression (25.0% +/- 6.4% to 67.9% +/- 5.0% 60 minutes after reperfusion), and increased ET-1 level in the portal blood (3.4 +/- 0.9 to 23.6 +/- 6.1 pg/mL). FK506 suppressed ET-1 expression (27.3% +/- 5.2%, 4.1 +/- 2.2 pg/mL), maintained microcirculation (96% +/- 16%), and blood pressure, reduced histologic damage, and improved survival. CsA had a similar but weaker effect compared with FK506. An additional experiment was performed with BQ485Na (BQ), an ETA receptor antagonist, to evaluate the genuine role of ET-1. BQ showed almost the same effects as FK506.. FK506 and CsA, particularly the former, maintain microcirculation and protect the tissue from I/R injury by suppressing the production and release of ET-1.

Topics: Animals; Azepines; Blood Pressure; Cyclosporine; Endothelin Receptor Antagonists; Endothelin-1; Hypotension; Immunosuppressive Agents; Intestine, Small; Ischemia; Male; Microcirculation; Oligopeptides; Portal System; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptors, Endothelin; Reperfusion Injury; Survival Rate; Tacrolimus; Time Factors

The neuroprotective properties of drugs binding to FKBP12, with and without subsequent inhibition of calcineurin, were investigated in rat models of ischemic embolic stroke. Drug effects on brain infarct volumes evoked by transient middle cerebral artery occlusion (MCAO) and by permanent MCAO were determined in vivo by T2-weighted magnetic resonance imaging and post mortem by triphenyltetrazolium chloride staining and histology. Drugs binding to FKBP12 and inhibiting calcineurin, such as FK506 and SDZ ASM 981, dose dependently reduced the infarct volumes, determined 48 h after MCAO by both magnetic resonance imaging and triphenyltetrazolium chloride staining but only in the transient MCAO model. In vivo potencies to reduce brain infarcts paralleled the in vitro potencies to inhibit calcineurin. Histological staining after 6 days of survival showed that the neuroprotective effects were permanent. Rapamycin, known to bind with similar affinity to FKBP12 but not to inhibit calcineurin, was not neuroprotective but abolished the neuroprotective effects of FK506 when coadministered. In the permanent MCAO models, FK506 showed no effect when injected before and little effect when injected after MCAO. Measurements of core temperatures after MCAO in controls and drug-treated rats do not support hypothermia being the mechanism responsible for neuroprotection. We conclude that drugs inhibiting calcineurin activity are neuroprotective in focal cerebral ischemia/reperfusion but not in permanent ischemia models, possibly by preventing reperfusion injury.

Topics: Animals; Arterial Occlusive Diseases; Calcineurin Inhibitors; Cerebral Arteries; Cerebral Infarction; Dose-Response Relationship, Drug; Enzyme Inhibitors; Immunosuppressive Agents; Ischemic Attack, Transient; Magnetic Resonance Imaging; Male; Neuroprotective Agents; Rats; Rats, Inbred SHR; Reperfusion Injury; Tacrolimus

Ceramide is a key mediator of apoptosis during the cellular stress response which is also involved in stroke-induced death. Transient occlusion of the middle cerebral artery (MCA) in rats led to a strong generation of ceramide as measured in thalamus and entorhinal cortex of the ischemic brain tissue. Enhanced levels of ceramide may be involved in apoptosis signaling following stroke since exogenously added synthetic C2-ceramide increased expression of c-jun and the death-inducing ligands (DILs) CD95-L, TRAIL and TNF-alpha in neuroblastoma cells. DILs in turn mediated death via binding to their respective receptors as concluded from diminished apoptosis upon blocking of the common pathway by dominant negative FADD. C2-ceramide induced both necrosis and apoptosis in a concentration-dependent manner corresponding to the situation present in the ischemic brain. The immunosuppressant FK506 inhibited the release of ceramide, expression of CD95-L and apoptosis in an in vitro and in vivo model for ischemia/reperfusion. These data suggest that ceramide is a crucial initiator of death, e.g., by induction of DILs following stroke.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain Chemistry; Cerebrovascular Disorders; Enzyme Inhibitors; Fas Ligand Protein; Gene Expression; Humans; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Membrane Glycoproteins; Necrosis; Neuroblastoma; Neurons; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sphingosine; Tacrolimus; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

Previous studies have demonstrated that the immunosuppressant FK506 provides neuroprotection in experimental brain injury and suggest that this action may be mediated by suppression of neuronal nitric oxide synthase activation that occurs after ischemic depolarization. We sought to determine whether FK506 reduces histological injury after middle cerebral artery occlusion (MCAO) in the rat and whether the neuroprotective effect is mediated via suppression of in vivo nitric oxide (NO) production during ischemia or early reperfusion.. Under controlled conditions of normoxia, normocarbia, and normothermia, halothane-anesthetized male Wistar rats were subjected to 2 hours of MCAO by the intraluminal occlusion technique in a blinded, randomized experimental trial. Ipsilateral parietal cortical laser-Doppler flowmetry was monitored throughout ischemia. Animals were randomly assigned to 4 pretreatment groups: intravenous FK506 0.3 mg/kg or 1. 0 mg/kg, vehicle (cremaphor), or an equivalent volume of saline administered 30 minutes before MCAO. Infarction volume was assessed by a triphenyltetrazolium chloride staining at 22 hours of reperfusion. In separate experiments, microdialysis probes were placed bilaterally into the striatum. Rats were perfused with artificial cerebrospinal fluid containing 3 micromol/L [14C]- L-arginine for 3 hours and then subjected to 2 hours of right MCAO. Intravenous 0.3 mg/kg FK506 or cremaphor was given 30 minutes before right MCAO. Right-left differences between [14C]-L-citrulline in the effluent were assumed to reflect differences in NO production.. All values are mean+/-SE. FK506 at 0.3 mg/kg reduced infarction volume in cortex: 40+/-12 mm3 compared with saline (109+/-15 mm3) and cremaphor vehicle (148+/-23) (P<0.05). Striatal infarction was also reduced by low-dose FK506: 16+/-4 mm3 versus 36+/-4 mm3 and 34+/-4 mm3 in saline and vehicle groups, respectively (P<0.05). High-dose treatment reduced infarction volume in cortex (61+/-14 mm3, P<0.05 from saline and vehicle groups) and in striatum (22+/-5 mm3, P<0.05 from saline and vehicle groups). [14C]-L-citrulline recovery via microdialysis was markedly enhanced in ischemic compared with nonischemic striatum. However, ischemia-evoked [14C]-L-citrulline recovery was not different in FK506-treated rats compared with vehicle-treated animals.. These data demonstrate that FK506 provides robust neuroprotection against transient focal cerebral ischemia in the rat. The mechanism of protection in vivo is not through attenuation of ischemia-evoked NO production during MCAO and early reperfusion.

Topics: Animals; Cerebral Infarction; Corpus Striatum; Ischemic Attack, Transient; Male; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Rats; Rats, Wistar; Reperfusion Injury; Tacrolimus; Time Factors

Neutrophils may play an important role in the development of liver ischemia/reperfusion injury. We investigated the effects of the immunosuppressants azathioprine (AZA), cyclosporine A (CsA), tacrolimus (FK506), and rapamycin (RPM) on the expression of cytokine-induced neutrophil chemoattractant (CINC) after ischemia/reperfusion of the liver.. Liver ischemia was induced in male Wistar rats by occluding the portal vein with a microvascular clip for 30 minutes. Rats received two intramuscular injections of AZA (4 mg/kg), CsA (5 mg/kg), FK506 (0.5 mg/kg), or RPM (0.5 mg/kg) 3 and 24 hours before ischemia/reperfusion of the liver.. Serum CINC concentrations in untreated animals increased, peaked 6 hours after reperfusion, and thereafter decreased gradually. Pretreatment with AZA, CsA, FK506, and RPM, however, inhibited the increase in serum CINC concentrations after reperfusion. CINC mRNA in liver tissue increased and peaked 3 hours after reperfusion, but was significantly lower in animals treated with AZA, CsA, FK506, and RPM. In vitro CINC production by Kupffer cells harvested from animals treated with AZA, CsA, FK506, or RPM 3 hours after reperfusion was also significantly lower than that observed in untreated animals. Both myeloperoxidase activity and the number of neutrophils accumulating in the liver 24 hours after reperfusion in animals treated with AZA, CsA, FK506, and RPM were significantly lower than in untreated animals. This correlated with lower serum aspartate transaminase, alanine transaminase, and lactate dehydrogenase levels in animals treated with AZA, CsA, FK506, and RPM 24 hours after reperfusion.. The immunosuppressants AZA, CsA, FK506, and RPM reduce neutrophil accumulation and attenuate ischemia/reperfusion injury of the liver.

Topics: Animals; Azathioprine; Cyclosporine; Drug Evaluation, Preclinical; Immunosuppressive Agents; Interleukin-16; Liver; Male; Neutrophils; Polyenes; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; Tacrolimus; Time Factors

The Ca2+/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca2+-containing medium after exposure to Ca2+-free medium caused Ca2+ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca2+-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca2+ challenge-induced injury in a dose-dependent manner (10(-10)-10(-8) M). Cyclosporin A at 1 microM mimicked the effect of FK506. Rapamycin (1 microM) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 nM), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca2+ challenge-induced increase in cytosolic Ca2+ level nor Na+-Ca2+ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca2+ level. Immunochemical studies showed that both calcineurin A (probably the A beta2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.

Topics: Animals; Astrocytes; Calcineurin; Calcium; Cells, Cultured; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Increased production of oxygen free radicals and infiltration of neutrophils into tissue subjected to ischemia-reperfusion have emphasized that neutrophils play a direct role in the development of injury. The present study was designed to elucidate the effect of FK506, a new immunosuppressive drug, on 11 hours of complete ischemia and reperfusion of the inguinal island skin flaps in rats. Group 1 (n = 10) control animals underwent ischemia and reperfusion and no treatment. Group 2 (n = 10) animals received FK 506 0.3 mg/kg/day, and group 3 (n = 9) animals received 0.5 mg/kg/day intramuscularly for 3 days before the ischemia. The effect of the drug was evaluated by measuring flap survival and tissue malondialdehyde content and myeloperoxidase activity and also by histopathologic examination of the skin specimens taken at the 1st and 24th hour after reperfusion. The survival of flaps controlled for 7 days was found to be significantly improved in group 2 (65.0 +/- 10.93 percent) and group 3 (93 +/- 6.25 percent) when compared with the control group (14 +/- 10.12 percent) (p < 0.04 and p < 0.0001). The tissue contents of malondialdehyde and activities of myeloperoxidase were significantly lower in groups 2 and 3 than in the control group. Three days of pretreatment with FK506 significantly reduced neutrophil infiltration in groups treated with either of the doses. These results showed that neutrophils play an important role in island flap survival associated with ischemia-reperfusion injury. Increased neutrophil infiltration was found related with increased levels of malondialdehyde and myeloperoxidase. Flap necrosis and the increase in malondialdehyde, myeloperoxidase, and neutrophil infiltration were improved by FK506 pretreatment, a neutrophil modulating agent.

Topics: Animals; Free Radicals; Graft Survival; Groin; Immunosuppressive Agents; Injections, Intramuscular; Ischemia; Leukocyte Count; Male; Malondialdehyde; Neutrophil Activation; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Skin; Skin Transplantation; Surgical Flaps; Tacrolimus

Topics: Animals; Immunosuppressive Agents; Ischemia; Liver Circulation; Reperfusion Injury; Tacrolimus

Liver ischemia and reperfusion injury is associated with activation of multiple inflammatory pathways, including free radicals, cytokines, and neutrophil-mediated tissue damage among others. Tacrolimus (FK506) has shown important regulatory effects on some inflammatory pathways, such as cytokines, neutrophils, and adhesion molecules. In this study, we explored a new potential protective mechanism for tacrolimus in the liver inflammatory response after ischemia and reperfusion, specifically its effect on liver tissue free radicals.. Total hepatic ischemia was produced in the rat for 90 min with an extracorporeal portosystemic shunt. Animals (n=96) were divided into four groups: group 1 comprised normal rats for reference values; group 2 comprised sham operated rats; in group 3, ischemic control rats received only the vehicle; and the experimental treatment group, group 4, received tacrolimus at a dose of 0.3 mg/kg, 4 hr before ischemia. Animal survival was followed up to 7 days. Liver function tests were performed and liver tissue free radicals and myeloperoxidase, serum cytokines (interleukin 1, tumor necrosis factor-alpha), and liver histology were measured 4 hr after reperfusion.. Seven-day survival was significantly improved from only 20% in the control group to 55% in the tacrolimus group (P<0.01). Liver function tests, histology, and myeloperoxidase tissue values were significantly improved (P<0.05) with tacrolimus pretreatment. Furthermore, a significant (P<0.05) down-regulation of serum cytokines and liver tissue free radicals was observed.. These data indicate a new and different protective mechanism for FK506 in regard to its ability to down-regulate free radical levels in livers subjected to severe ischemia and reperfusion. Tacrolimus, also confirmed to be a potent suppressor of the cytokine response, specifically interleukin 1 and tumor necrosis, decreased neutrophil tissue migration as well.

Topics: Animals; Cytokines; Down-Regulation; Free Radicals; Immunosuppressive Agents; Interleukin-1; Liver; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus; Tumor Necrosis Factor-alpha

Increased morbidity and mortality following transplantation surgery due to the primary nonfunction and dysfunction of the liver poses a great challenge and has increased the crescendo of research work in this field. In this study, we have tried to address the issue concerning the changes in Ca2+ homeostasis and hepatic microcirculation in 90 min of ischemia followed by reperfusion of the liver after FK506 pretreatment. Twenty dogs divided into two groups; group I (0.15 mg/kg/day FK506 for 3 days, im) and group II (control) were used for the measurement of mitochondrial (mit) and total cellular Ca2+ by atomic absorption spectrophotometer and hepatic microcirculation with laser Doppler flowmeter. Serum AST leakage was significantly (P < 0.05) suppressed in group I at 6 hr after reperfusion. The percentage gain in mit Ca2+ in group I was significantly (P < 0.05) inhibited compared to that in group II at 15 min after reperfusion and also when compared with the preischemic value it was significantly (P < 0.05) elevated at 30 min after reperfusion in group II only. FK pretreatment significantly (P < 0.05) inhibited the overload in total cellular Ca2+ at 15 and 30 min after reperfusion. Moreover, hepatic microcirculation was significantly (P < 0.001) better in group I between 2 and 6 hr after reperfusion. In conclusion, the ameliorating property of FK in ischemia-reperfusion may be explained by prevention of the cellular Ca2+ overload during the perireperfusion period.

Topics: Animals; Calcium; Dogs; Female; Homeostasis; Immunosuppressive Agents; Ischemia; Liver; Liver Circulation; Male; Microcirculation; Reperfusion Injury; Tacrolimus

Topics: Alanine Transaminase; Analysis of Variance; Animals; Cold Temperature; Graft Survival; Ischemia; Lipid Peroxides; Liver; Liver Transplantation; Organ Preservation; Phospholipids; Rats; Rats, Inbred ACI; Reperfusion Injury; Tacrolimus; Transplantation, Isogeneic

We examined whether an immunosuppressant, FK506, inhibits delayed neuronal death in the gerbil hippocampal CA1 sector after 5-min forebrain ischemia. After reperfusion, gerbils were injected intravenously with FK506. Gerbils in the early injection group were injected with FK506 immediately after reperfusion, and gerbils in the delayed injection group were injected with FK506 1 or 2 h postischemia. The body temperature of the FK506-treated gerbils in the normothermic group was maintained at 37.5-38.0 degrees C for 2 h postischemia. In the chronic survival group, neuroprotection was assessed after recovery for 45 days. Seven or 45 days after reperfusion, neuronal density in the CA1 was assessed following perfusion fixation. FK506 ameliorated cell death in the CA1 in a dose-dependent manner in every group, although it showed a hypothermic effect. FK506 is neuroprotective against forebrain ischemia in gerbils.

Topics: Animals; Body Weight; Cell Death; Dose-Response Relationship, Drug; Gerbillinae; Hippocampus; Hypothermia; Immunosuppressive Agents; Male; Neurons; Neuroprotective Agents; Prosencephalon; Reperfusion Injury; Tacrolimus; Time Factors

Topics: Adenosine; Allopurinol; Animals; Cell Adhesion; Cold Temperature; Endothelium, Vascular; Glutathione; Graft Survival; Insulin; Ischemia; Leukocytes; Liver Circulation; Liver Transplantation; Microcirculation; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred ACI; Reperfusion Injury; Tacrolimus; Transplantation, Isogeneic

Reperfusion after ischemia results in endothelial cell injury and Kupffer cell activation. Inflammatory cytokines thus released can induce major histocompatibility complex antigens and increase the immunogenecity of the graft. An orthotopic rat liver allotransplant model was used to test the hypothesis that prevention of reperfusion injury by infusion of polyethylene glycol superoxide dismutase (PEG-SOD) would result in long-term allograft survival in the presence of subthreshold immunosuppressive dosages. ACI rats were used as donors, and Lewis strain rats as recipients. Orthotopic liver transplantation was initially performed to identify a subthreshold dose of the immunosuppressant FK-506, which would be unable to extend survival longer than control untreated rats with this strain combination. After testing three intramuscular FK-506 doses of 0.04, 0.08, and 0.16 mg/kg, it was observed that an FK-506 dose of 0.04 mg/kg/day for 14 days was unable to extend survival longer than in untreated recipients. This dose of FK-506 was used in combination with PEG-SOD at doses of 1000, 3000, 10,000, or 30,000 units. Recipient animals were treated intravenously with PEG-SOD as a loading dose to facilitate tissue penetration on day 1, and beginning on the day of transplantation, every 2 days for the duration of the study. Results of histologic studies and mean survival time were compared in untreated recipients and in rats treated with PEG-SOD plus 0.04 mg/kg/day FK-506.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Drug Interactions; Graft Rejection; Graft Survival; Liver; Liver Transplantation; Polyethylene Glycols; Rats; Rats, Inbred ACI; Rats, Inbred Lew; Reperfusion Injury; Superoxide Dismutase; Tacrolimus; Transplantation, Homologous

In order to study further whether a relationship exists between the extent of ischemia-preservation-reperfusion injury (IPRI) and acute rejection (AR) events in liver allografts, we retrospectively reviewed 213 consecutive cyclosporine-treated patients who received their first liver allograft between 1/1/93 and 12/31/93. Of these, 178 fulfilled the study inclusion criteria. The extent of IPRI was assessed by the peak value of aspartate aminotransferase (ASTmax) observed within the first 72 hr posttransplant. For the purpose of univariate analysis, categorical classification of recipients was done based upon ASTmax as follows: group 1, ASTmax < 600 IU/L (n = 43); group 2, ASTmax 600-2000 IU/L (n = 86); and group 3, ASTmax > 2000 IU/L (n = 49). For multivariate analysis, stepwise Cox regression was performed with age, ASTmax, and UNOS status as covariates. At a median follow-up of 271 days there were no statistically significant differences between groups with respect to the incidence of a first episode of AR (47%, 55%, 51%, respectively, P = NS), the timing of AR (respective medians, 9, 10, and 10 days, P = NS), or the proportion of patients treated with OKT3 (9%, 20%, 12%, respectively, P = NS) or converted to FK506 (16%, 12%, 10%, P = NS). Cox regression confirmed the lack of an independent association between the extent of IPRI and any of these outcomes. We conclude that in UW-preserved, cyclosporine-treated primary liver allografts, no correlation exists between the extent of IPRI and the incidence, timing, severity, or refractoriness of clinically defined AR events.

Topics: Cyclosporine; Graft Rejection; Graft Survival; Humans; Immunosuppression Therapy; Liver Transplantation; Muromonab-CD3; Organ Preservation; Reperfusion Injury; Retrospective Studies; Tacrolimus

The mechanism by which FK506 (FK) prevents hepatic injury induced by ischemia/reperfusion was studied. Adult Sprague-Dawley rats were subjected to 60-min normothermic liver ischemia. Animals were divided into two groups: group I, controls, saline vehicle treatment; group II, FK treatment. FK (1 mg/kg/day, p.o.) was given for 4 consecutive days prior to inducing ischemia. In addition to a survival study, plasma levels of endotoxin and serum activities of tumor necrosis factor-alpha (TNF) and aspartate aminotransferase (AST) were assessed in the blood collected from suprahepatic vena cava. Results showed: (1) FK therapy significantly improved 7-day survival (80.0%) compared with nontreated animals (50.0%, p < 0.05); (2) both TNF and endotoxin were elevated following reperfusion, reaching maximum values at 3 h after reperfusion (217.0 +/- 40.6 and 280.5 +/- 31.4 pg/ml, respectively, in the control; mean +/- SEM), and (3) serum activities of TNF and AST following reperfusion were substantially suppressed with FK treatment, whereas FK did not reduce the rise in endotoxin. These findings suggest that suppression of TNF production in response to endotoxemia might account at least in part for the protective effect of FK against ischemia-induced hepatic injury.

Topics: Animals; Aspartate Aminotransferases; Endotoxins; Female; Liver; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Survival Analysis; Tacrolimus; Tumor Necrosis Factor-alpha

The mechanisms by which polymorphonuclear neutrophils (PMNs) are recruited by the ischemic and reperfused liver are still unknown. The purpose of this study was to determine whether tumor necrosis factor-alpha (TNF) and/or interleukin-1 alpha (IL-1) acted as potential mediators for PMN infiltration after liver ischemia and reperfusion. The potential effect of FK 506, a powerful immunosuppressant, was also studied. Male Sprague-Dawley rats were subjected to 60 and 90 min of total hepatic ischemia, with an extracorporeal porto-systemic shunt. FK 506 (0.3 mg/kg) was intravenously administered 4 hr before ischemia (FK 506 group), and control animals received normal saline solution (NS group). Plasma TNF, IL-1 levels, and PMN infiltration in liver tissue were serially examined at the end of ischemia, 5, 30, 60, and 360 min after reperfusion. The degree of liver necrosis was assessed at 360 min following reperfusion. In the NS group, IL-1 and TNF revealed a transient elevation at 30 and 60 min after reperfusion, following 60 min of ischemia. When the ischemia was increased to 90 min, the IL-1 activity had a rapid elevation (330.5 +/- 129 pg/ml) at 5 min, which remained at high levels (197.8 +/- 70.4 pg/ml) until 6 hr after reperfusion, whereas the TNF activity decreased to normal levels following a similar peak (355 +/- 181.9 pg/ml) at 5 min after reperfusion. The time course of IL-1 release in the NS group, with 90 min of ischemia, correlated directly with the PMN infiltration.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Chemotaxis, Leukocyte; Interleukin-1; Ischemia; Liver; Male; Neutrophils; Portasystemic Shunt, Surgical; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus; Tumor Necrosis Factor-alpha

Topics: Adenosine Triphosphate; Animals; Bile; Cold Temperature; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Circulation; Male; Organ Preservation; Portal System; Rats; Rats, Inbred Lew; Regional Blood Flow; Reperfusion Injury; Tacrolimus

Topics: Animals; Graft Survival; Ischemia; Liver; Liver Circulation; Liver Function Tests; Liver Transplantation; Organ Preservation; Regional Blood Flow; Reperfusion Injury; Swine; Tacrolimus; Temperature

The protective effect of FK506 on hepatocytes against ischemia and reperfusion injury was examined by evaluating the following: the high energy phosphorus metabolism obtained using 31P magnetic resonance spectroscopy (31P-MRS) and the tissue blood flow of the liver in ischemia and the reperfusion process, mitochondrial glutamic oxaloacetic transaminase (m-GOT) and glutamic pyruvic transaminase (GPT), the survival rates of the animals, a histological study and immunohistological staining for intercellular adhesion molecule-1 (ICAM-1) in the liver after ischemia. The rats were treated with FK506 1 mg/kg/day i.m. for 4 days before testing. Ischemia was induced by clamping the hepatoduodenal ligament for 30 min. In 31P-MRS, the recovery of the hepatic energy status after ischemia, evaluated by beta-ATP/inorganic phosphate (Pi), was significantly better in the FK506 group. It also coincided with the recovery of tissue blood flow monitored with a laser Doppler flowmeter. In the histological examination, the congestion observed in the periportal region of the control group was mild, while there was less induction of ICAM-1 in the endothelial cells of the portal veins and hepatic veins in the FK506 group. From these findings, we concluded that FK506 had a protective effect on hepatocytes against warm ischemia and reperfusion injury, and the mechanism for this could partially be attributed to improved tissue blood flow after ischemia by the modulation of immunological events.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Blood Flow Velocity; Immunohistochemistry; Intercellular Adhesion Molecule-1; Liver; Magnetic Resonance Spectroscopy; Male; Mitochondria, Liver; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Mechanisms by which immunodepressants (Cyclosporine, CsA; FK 506, FK; Azanthioprine, AZA) ameliorate warm ischemic injury of the liver were examined. Female Sprague-Dawley rats were subjected to 60-min of normothermic liver ischemia. Animals were assigned to one of four groups: group I, control with vehicle treatment; groups II, III, and IV, treatment with CsA (10 mg/kg), FK (1 mg/kg), and AZA (1 mg/kg), respectively. The immunosuppressive agents were given per os for 4 consecutive days prior to the induction of hepatic ischemia. In addition to a survival study, plasma levels of endotoxin, serum activities of tumor necrosis factor-alpha (TNF), plasma levels of phosphatidylcholine hydroperoxide (PCOOH) as a lipid peroxide, and serum alanine aminotransferase (ALT) were investigated in blood samples collected from the suprahepatic vena cava. A 7-day survival period was significantly higher in the immunosuppressed animals. Serum TNF levels were elevated and peaked at 3 h following reperfusion. When, the peak values were compared, the animals given immunodepressants had significantly lower levels of TNF (217.0 +/- 40.6 pg/ml for group I, 67.6 +/- 13.7 for group II, 87.9 +/- 28.3 for group III and 89.1 +/- 19.9 for group IV; Mean +/- SEM). Plasma PCOOH levels were also elevated following reperfusion, but with no statistical difference among the groups. Our data suggest that immunodepressants ameliorate warm ischemia/reperfusion injury through modulation of TNF production and not through a diminution of lipid peroxidative injury.

Topics: Alanine Transaminase; Animals; Azathioprine; Cyclosporine; Female; Immunosuppressive Agents; Ischemia; Lipid Peroxidation; Liver; Phosphatidylcholines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus; Temperature; Time Factors; Tumor Necrosis Factor-alpha

Topics: Adenosine Triphosphate; Alanine Transaminase; Animals; Aspartate Aminotransferases; Cyclosporine; Dogs; Female; Ischemia; L-Lactate Dehydrogenase; Liver; Male; Reperfusion Injury; Tacrolimus

To examine the role of neutrophils, their presence and the degree of infiltration, as important determinants of ischemia and reperfusion injury of the liver, male Sprague-Dawley rats were subjected to 60 and 90 min of total-liver ischemia. The presence of neutrophils, assessed by the measurement of liver tissue myeloperoxidase (MPO), and the degree of neutrophil liver infiltration, determined by the naphthol AS-D chloroacetate esterase technique, correlated well with animal survival and response to FK506 and cyclosporine administration. Lipid peroxidation, measured by the malondialdehyde (MDA) test in liver tissue, was another factor closely linked with liver function and survival. Pretreatment with FK506 (0.3 mg/kg) and CsA (5 mg/kg) was given at 4 hr and 1 hr before ischemia and at the time of reperfusion. Control ischemic animals showed increased neutrophil liver infiltration, high MPO and MDA liver levels, and diminished overall survival. FK506 and CsA-treated animals had better survival and diminished neutrophil liver infiltration, as well as MPO and MDA levels. The mechanism by which FK506 and CsA protected the animals from severe liver ischemic injury is unknown. Our data indicated that the presence and the degree of infiltration of neutrophils were important components of liver ischemia/reperfusion injury in the rat. So it is possible that one of the fundamental effects of the FK506 and CsA might be through the inhibition of the presence and infiltration of neutrophils in liver tissue.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cyclosporine; Ischemia; L-Lactate Dehydrogenase; Liver; Male; Malondialdehyde; Microscopy, Electron; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Survival Analysis; Tacrolimus

Using a rat model, the effect of pre-treatment with FK 506 on hepatic ischemia/reperfusion injury was investigated. All control animals died within 72 h of the ischemia/reperfusion injury. Pre-treatment of the animals with FK 506 (0.3 mg/kg in 0.5 ml saline) administered intravenously improved survival. The most striking protection against fatal ischemia/reperfusion injury was achieved in rats that were given FK 506 6 and 24 h prior to the induction of the hepatic ischemic insult (70% and 80% 10-day survival rates, respectively). The hepatoprotective effect of FK 506 was assessed further in a second experiment in which the serum levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6) were measured. These results suggest that a 60-min period of hepatic ischemia and subsequent reperfusion triggers the release of both TNF and IL-6, and that FK 506 pre-treatment (6 h before the ischemic episode) significantly inhibits the production and/or release of these two cytokines compared to untreated controls. These data provide additional information concerning the immunosuppressive and hepatoprotective activities of FK 506. Based upon these data, it is probable that FK 506 attenuates hepatic ischemia/reperfusion injury, at least in part, by reducing TNF and IL-6 levels.

Topics: Animals; Hepatectomy; Interleukin-6; Liver; Male; Rats; Rats, Inbred Lew; Reperfusion Injury; Tacrolimus; Tumor Necrosis Factor-alpha

The effect of pretreatment with FK506 on renal ischemia and reperfusion (I/R) injury was investigated using a rat model. Animals were assigned to one of two groups (20 rats each). Group 1 animals (controls) received 0.5 ml saline while group 2 animals received FK506 (0.3 mg/kg), administered intravenously 24 hr prior to the induction of renal ischemia. A 60-min period of ischemia of the right kidney was induced, and upon reperfusion a left nephrectomy was performed. Blood samples for estimation of BUN, creatinine, and tumor necrosis factor were collected on days 0 (preischemia), 1, 2, 3, 5, 7, and 10 (postischemia). Rats were sacrificed after day 10 and renal tissue was examined histologically. All animals survived the ischemic episode. FK506 pretreatment significantly reduced the serum levels of BUN (P less than 0.02), creatinine (P less than 0.02), and TNF (P less than 0.05) as compared with that seen in controls. Histologically, at day 10, the kidneys showed the expected sequelae of prior renal I/R with various degrees of tubular damage. However, no objective differences were evident between the two groups. Based upon these data, it can be concluded that (1) FK506 pretreatment ameliorates the functional renal injury associated with I/R, (2) renal ischemia induces the release of TNF, and (3) FK506 pretreatment results in a significant inhibition of TNF production. These data suggest that the release of TNF may be responsible for the increasing of BUN and creatinine levels seen after renal I/R and that pretreatment of renal donors with FK506 may improve renal function in the immediate post-transplant period.

Topics: Animals; Kidney; Kidney Function Tests; Kidney Tubules; Male; Premedication; Rats; Rats, Inbred Lew; Reperfusion Injury; Tacrolimus

The effect of FK 506 pretreatment on renal ischemia and reperfusion (I/R) injury was investigated. Adult male rats were assigned to one of two groups (20 animals each). Group 1 (controls) received 0.5 mL saline while group 2 received FK 506 (0.3 mg/kg) intravenously 24 h prior to the induction of renal ischemia. After a 60-min period of ischemia of the right kidney, a left nephrectomy was performed. Blood for BUN, creatinine, and tumor necrosis factor (TNF) was obtained prior to ischemia and on days 1, 2, 3, 5, 7, and 10. All surviving animals were sacrificed at day 10. FK 506 pretreatment reduced the serum levels of BUN (p less than .02), creatinine (p less than .02) and TNF (p less than .05) as compared to that seen in controls. Based upon these data, it appears that: (a) renal ischemia induces the release of TNF; (b) FK 506 pretreatment inhibits TNF production; and (c) FK 506 reduces renal injury association with I/R.

Topics: Animals; Blood Urea Nitrogen; Creatinine; Kidney; Male; Rats; Rats, Inbred Lew; Reperfusion Injury; Tacrolimus; Time Factors; Tumor Necrosis Factor-alpha

The present study was designed to elucidate the effect of FK506 on 90 min of warm ischemia of the liver and reperfusion in 30 dogs. Three groups of animals were studied. Group 1 animals received FK (0.15 mg/kg/day) for three days prior to the ischemia and group 2 animals got 2 ml of saline solution for three days instead of FK and were considered controls. In group 3 FK (0.15 mg/kg/day) was injected immediately upon reperfusion and two days thereafter. Evaluation of the effectiveness of the drug was monitored by measuring the serum activities of AST, ALT, LDH, serum total bilirubin, malondialdehyde, and by histopathological examinations of the liver specimens and survival of the animals for 7 days after reperfusion. The 7 day survival of the animals in group 1 (80%) was significantly (P < 0.05) improved compared with those in group 2 (30%) and group 3 (20%). The serum activities of AST, ALT, and LDH and total bilirubin were significantly lower in group 1 than in group 2 and group 3. FK pretreatment significantly prevented hepatocellular necrosis and neutrophilic infiltration in group 1 in comparison with those in group 2 and group 3. Although the malondialdehyde level in hepatic venous blood was relatively lower in group 1, this difference was not statistically significant. Three days FK pretreatment prevented hepatocellular injury and enzyme leakage after 90 min of hepatic ischemia, whereas FK treatment immediately upon reperfusion failed to do so. In conclusion, donor organ pretreatment with FK may become a promising strategy for improved allograft survival in liver transplantation.

Topics: Animals; Dogs; Female; Hepatic Veins; Liver; Male; Malondialdehyde; Premedication; Reperfusion Injury; Tacrolimus

The effect of FK 506 on regeneration of the liver was studied in rats after a two-thirds partial hepatectomy after 60 min of ischemia of the unresected liver. The animals were divided into three distinct groups of 10 rats each. Group 1 (controls) received 0.5 ml saline solution intravenously 30 min after the induction of ischemia. Groups 2 and 3 were injected with FK 506 (0.3 mg/kg) intravenously 30 min after and 24 min before the induction of hepatic ischemia, respectively. The hepatic content of ATP and serum levels of ALT and lactate dehydrogenase were determined on each animal. In addition, the histological appearance and mitotic activity of the remnant liver was determined at regular 24-hr intervals after hepatic ischemia. All 10 control animals died within 72 hr. Treatment with FK 506 resulted in improved survival in groups 2 and 3 (30% and 80%, respectively). The improved survival seen in the FK 506-treated animals was reflected by a restoration of hepatic ATP content, a reduction in the serum levels of ALT and lactate dehydrogenase, an amelioration of hepatic necrosis and neutrophilic infiltration and an increase in the mitotic activity of the liver. These results suggest that FK 506 ameliorates the hepatic injury associated with ischemia/reperfusion and has a potent stimulatory effect on liver cell regeneration that may make it valuable as a hepatoprotective agent when administered to organ donors before graft harvesting.

Topics: Adenosine Triphosphate; Alanine Transaminase; Animals; Anti-Bacterial Agents; Immunosuppressive Agents; L-Lactate Dehydrogenase; Liver; Liver Diseases; Liver Regeneration; Male; Mitosis; Necrosis; Rats; Rats, Inbred Lew; Reperfusion Injury; Tacrolimus

Isolated segments of cat small intestine were subjected to 3 hr of ischemia followed by 1 hr reperfusion (I/R). Mucosal biopsies were obtained for measurement of myeloperoxidase (MPO) activity, an index of tissue neutrophil count, and leukotriene B4 (LTB4) production. Animals were pretreated with either cyclosporin A (CsA) or FK506, which are potent immunosuppressants. Both agents significantly attenuated the neutrophil infiltration induced by I/R. FK506, but not CsA, reduced the elevated mucosal LTB4 production normally observed following reperfusion. The results of this study suggest that FK506 and CsA may be important agents in modulating neutrophil infiltration in acute inflammatory conditions.

Topics: Animals; Cats; Cyclosporine; In Vitro Techniques; Intestine, Small; Ischemia; Leukocyte Count; Leukotriene B4; Neutrophils; Peroxidase; Reperfusion Injury; Tacrolimus

Ischemic damage of the allograft liver is a major problem in clinical liver transplantation. Therefore the identification of hepatoprotective agents is a high priority at most liver transplantation programs. FK 506, a potent new immunosuppressive agent has been reported to possess hepatotrophic activity. To evaluate the putative hepatotrophic activity of FK 506 on experimental hepatic ischemia, rats were subjected to a subtotal hepatectomy following experimental ischemia and subsequent rat survival was assessed. FK 506 (0.3 mg/Kg) administered intravenously 24 hours prior to the induction of hepatic ischemia, reduced the subsequent mortality rate from 100% among controls given saline to only 20% (P less than 0.001). This observation demonstrates that FK 506 enhances the regenerative response of the liver to ischemic injury and may, in addition to its immunologic activity have hepatotrophic activity as well.

Topics: Animals; Anti-Bacterial Agents; Hepatectomy; Immunosuppressive Agents; In Vitro Techniques; Ischemia; Liver; Male; Premedication; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate; Tacrolimus