tacrolimus and Brain-Ischemia

Immunophilins consist of a family of highly conserved proteins binding with immunosuppressive drugs such as FK506, rapamycin and cyclosporin A. FK506-binding protein (FKBP) is one of two major immunophilins and most of FKBP family members bind FK506 and show peptidylprolyl cis/trans isomerase (PPIase) activity. Small size FKBP family members contain only FK506-binding domain, while FKBPs with large molecular weights possess extra domains such as tetratricopeptide repeat domains, calmodulin binding and transmembrane motifs. FKBPs are involved in several biochemical processes including protein folding, receptor signaling, protein trafficking and transcription. FKBP family proteins play important functional roles in the T-cell activation, when complexed with their ligands. The roles of immunophilins in protein transportation and apoptosis through their molecular interactions with receptors or proteins have emerged recently. Moreover, therapeutic implications of immunophilin ligands in treating neurodegenerative disorders have been accumulating. FK506 and its derivatives with no immunosuppressive activities bind to the conserved active sites of the canonical FKBP members such as FKBP12, which shows PPIase activity. These immunophilin ligands show variable efficacy in animal models for Parkinson's disease, dementia, and spinal cord injury, where the canonical immunophilins function as chaperones and are associate with the protein folding and modulation of oxidative stress. On the other hand, in the noncanonical FKBP members such as FKBP38, FK506-binding site is not conserved and shows neither PPIase activity nor affinity to FK506. Interestingly, the small molecule-mediated inhibition of the noncanonical member of FKBP family appears to cause neuronal protection and induce proliferation of neuronal stem cells in a rat focal cerebral ischemia model. Currently, the mechanisms of actions remain unclear. This review focuses on molecular characteristics of the canonical and noncanonical FKBP family members and the biological functions of their ligands in performing neuroprotective and neurotrophic activities.

Topics: Animals; Brain Ischemia; Humans; Immunophilins; Lymphocyte Activation; Models, Immunological; Models, Molecular; Molecular Chaperones; Nerve Tissue Proteins; Neurons; Oxidative Stress; Protein Binding; Protein Conformation; Protein Folding; Protein Interaction Mapping; Protein Structure, Tertiary; Rats; Stem Cells; Structure-Activity Relationship; T-Lymphocytes; Tacrolimus; Tacrolimus Binding Proteins

Recent advances in MRI technology and the development of effective neuroprotective agents has improved the outcome of stroke. In order to salvage tissue after an ischemic insult it is important to differentiate the core and penumbra area of the ischemic lesion. The penumbra surrounds the ischemic core, damage in this area is reversible if effective neuroprotective agents are given and reperfusion occurs. In this symposium detection of penumbra in an ischemic lesion using diffusion weighted imaging (DWI) and perfusion imaging (PI), diffusion-perfusion mismatch, and indications for thrombolytic therapy are discussed. If a hypoperfusion area is revealed with PI without a corresponding lesion indicated with DWI or when the DWI lesion is less than one third of the PI lesion, combined thrombolytic and neuroprotective therapies are recommended. In contrast, when both PI and DWI show an identical lesion, only neuroprotective therapy is advised. Additionally, newly developed neuroprotective agents, especially the combined effect of rt-PA and the immunosuppressant, FK506, on an embolic infarct model are discussed.

Topics: Acute-Phase Reaction; Antipyrine; Brain Ischemia; Cerebrovascular Circulation; Drug Design; Drug Therapy, Combination; Edaravone; Humans; Immunosuppressive Agents; Magnetic Resonance Imaging; Nerve Growth Factors; Neuroprotective Agents; Niacinamide; Recombinant Proteins; Tacrolimus; Time Factors; Tissue Plasminogen Activator

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

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

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

Cerebral ischemia in the territory of the middle cerebral artery (MCA) can induce delayed neuronal cell death in the ipsilateral substantia nigra (SN) remote from the primary ischemic lesion. This exofocal postischemic neuronal degeneration (EPND) may worsen stroke outcomes. However, the mechanisms leading to EPND are poorly understood. Here, we studied the time course of EPND via sequential magnetic resonance imaging (MRI) and immunohistochemistry for up to 28 days after 30 minutes' occlusion of the MCA (MCAo) and reperfusion in the mouse. Furthermore, the effects of delayed treatment with FK506 and MK-801 on the development of EPND were investigated. Secondary neuronal degeneration in the SN occurred within the first week after MCAo and was characterized by a marked neuronal cell loss on histology. Sequential neuroimaging examinations revealed transient MRI changes, which were detectable as early as day 4 after MCAo and thus heralding histologic evidence of EPND. Treatment with MK-801, an established anti-excitotoxic agent, conferred protection against EPND even when initiated days after the initial ischemic event, which was not evident with FK506. Our findings define a secondary time window for delayed neuroprotection after stroke, which may provide a promising target for the development of novel therapies.

Topics: Animals; Brain Ischemia; Cell Count; Dizocilpine Maleate; Immunohistochemistry; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Magnetic Resonance Imaging; Male; Mice; Mice, 129 Strain; Nerve Degeneration; Neuroprotective Agents; Substantia Nigra; Tacrolimus

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

The immunosuppressant drug rapamycin was reported to have an antiaging activity, which was attributed to the TORC1 inhibition that inhibits cell proliferation and increases autophagy. However, rapamycin also exhibits a number of harmful adverse effects. Whether rapamycin can be developed into an antiaging agent remains unclear.. We demonstrated that rapamycin at micro-doses (below the TORC1 inhibiting concentration) exhibits a cell-protective activity: (1) It protects cultured neurons against neurotoxin MPP(+) and H2O2. (2) It increases survival time of neuron in culture. (3) It maintains the nonproliferative state of cultured senescent human fibroblasts and prevents cell death induced by telomere dysfunction. (4) In animal models, it decreased the cerebral infarct sizes induced by acute ischemia and dramatically extended the life span of stroke prone spontaneously hypertensive rats (SHR-SPs).. We propose that rapamycin at micro-dose can be developed into an antiaging agent with a novel mechanism.

Topics: Aging; Animals; beta-Galactosidase; Brain Infarction; Brain Ischemia; Cell Death; Cell Line, Transformed; Cerebral Cortex; Disease Models, Animal; Fibroblasts; Humans; Hydrogen Peroxide; Immunosuppressive Agents; Male; Mice, Inbred C57BL; Phosphorylation; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Tacrolimus

We examined the protective effects of the immunosuppressants cyclosporin A (CsA) and FK506 on abnormal cytosolic Ca²⁺ ([Ca²⁺]c) and mitochondrial Ca²⁺ concentration ([Ca²⁺]m) dynamics induced by ischemia or high L-glutamate concentration in mouse brain slice preparations. We used fura-4F and rhod-2 as indicators for [Ca²⁺]c and [Ca²⁺]m, respectively, in their acetoxymethylester form. Slice preparations loaded with either of these two indicators were exposed to ischemic artificial cerebrospinal fluid (oxygen- and glucose-deprived medium) for 12 min or to aerobic medium with high L-glutamate concentration (isotonic 20 mM L-glutamate) for 5 min. CsA (1 - 10 μM) showed significant protective effects on the maximum increase in ischemia-induced [Ca²⁺]c and [Ca²⁺]m. FK506 (10 μM) showed significant protective effects on the [Ca²⁺]m increase, but not on the ischemia-induced [Ca²⁺]c increase. Both immunosuppressants showed almost equal protective effects on the [Ca²⁺]c and [Ca²⁺]m increases induced by high L-glutamate concentration. These results suggest that the protective effects of CsA and FK506 on Ca²⁺ overloading may be dependent upon the common pharmacological sites of actions relating to their effects as immunosuppressants. The small, but significant depressant effects of these drugs could give us important clues for rescuing critical brain damage induced by Ca²⁺ overloading.

Topics: Animals; Brain; Brain Edema; Brain Ischemia; Brain Mapping; CA1 Region, Hippocampal; Calcium Signaling; Cerebral Cortex; Cyclosporine; Cytosol; Glutamic Acid; Immunosuppressive Agents; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mitochondria; Neurons; Neuroprotective Agents; Osmolar Concentration; Tacrolimus

Transplantation of bone marrow stromal cells (MSCs) has been shown to ameliorate ischemic brain injury in animals. In the present study, we investigated whether the transplantation of MSCs combined with FK506, a clinically used immunosuppressant, enhanced neuroprotective effects in rat experimental stroke.. Male Sprague-Dawley rats underwent transient 90 min middle cerebral artery occlusion (MCAO). Two or 6h after ischemia onset, the rats were randomly assigned to receive intravenous administration of MSCs plus FK506, MSCs alone, FK506 alone, or vehicle. Infarct volume, and neurological and immunohistological assessments were performed to examine the effects of these therapies.. In 2-hour post-ischemia treatment groups, significant improvement of infarct volume and neurological scores were observed 1 day after combination therapy compared with monotherapy, and this neuroprotection continued for 7 days. Combination therapy significantly reduced the number of TUNEL-positive apoptotic cells, increased Bcl-2 expression, decreased Bax expression, and suppressed neutrophil infiltration and microglia/macrophage activation compared to monotherapy. In 6-hour post-ischemia treatment groups, a significant reduction of infarct volume, edema index, and neurological score was observed only in the combination therapy group. Moreover, the number of engrafted MSCs on day 7 with combination therapy was significantly higher than with MSCs alone.. Combination therapy using FK506 enhanced the anti-apoptotic and anti-inflammatory effects of MSCs and increased the survival of transplanted cells, leading to expansion of the therapeutic time window for MSCs.

Topics: Analysis of Variance; Animals; Apoptosis; bcl-2-Associated X Protein; Bone Marrow Transplantation; Brain Ischemia; Combined Modality Therapy; Immunohistochemistry; In Situ Nick-End Labeling; Male; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Stromal Cells; Tacrolimus; Treatment Outcome

The immunosuppressant FK506 (1 mg/kg, i.p.) reduces the infarct size following 90 min occlusion of the middle cerebral artery (MCAo) in adult rat brain. Here we have investigated the effect of FK506 on cerebral immune cells that are considered to contribute to neurodegeneration. FK506 substantially attenuated the response of resident and peripheral immune cells following transient ischemia. Between 24 hr and 5 days after MCAo, FK506 reduced the T-cell infiltration in the infarct area as well as the presence of activated and/or phagocytic OX-18, OX-42, GSA-IB4, Iba1, and ED1 positive microglia/macrophages. FK506 also lowered the protein levels of TNFalpha and IL-2 in ischemic brain areas. Repetitive application of FK506 over 20 days attenuated the activation of microglia in the substantia nigra (SN), an area of secondary degeneration. Importantly, FK506 conferred also lasting protection of the neurons of SN; these neurons degenerate by withdrawal of neurotrophic factors from the striatum that undergoes necrotic death as part of the ischemic core. To understand the molecular basis of FK506 effects in cerebral immune cells, we determined in primary postnatal day 0/1 (P0/P1) microglia (i) the expression of the FK506 binding proteins FKBP12, FKBP52, and FKPB65 and (ii) that FK506 (1-100 ng/mL) lowered the number of resting or lipopolysaccharide stimulated microglia as well as we induced the lipopolysaccharide release of TNFalpha in a dose-dependent manner. In summary, FK506 confers rescue of brain tissue following cerebral ischemia not only by neuronal protection, but also by suppression of microglial activation and peripheral immune responses.

Topics: Animals; Biomarkers; Brain Ischemia; Cells, Cultured; Cerebral Infarction; Chemotaxis, Leukocyte; Coculture Techniques; Corpus Striatum; Cytokines; Disease Models, Animal; Encephalitis; Gliosis; Immunosuppressive Agents; Macrophages; Male; Microglia; Nerve Degeneration; Nerve Tissue Proteins; Neuroprotective Agents; Protein Binding; Rats; Rats, Sprague-Dawley; Substantia Nigra; T-Lymphocytes; Tacrolimus

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemia-reperfusion injury caused by transient focal brain ischemia induced by middle cerebral artery (MCA) occlusion for 60 min in rats. Neuronal damage visualized as a decrease of MAP2 immunoreactivity was observed in the cerebral cortex at 9 h after MCA occlusion and further expanded at 24 h. Hypoxic areas visualized with an immunohistochemical reaction for 2-nitroimidazole, a hypoxia marker (hypoxyprobe-1), and accumulation of granulocytes and platelets were also observed at 9 h and 24 h after MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, attenuated cortical damage and decreased the hypoxyprobe-1 positive area, as well as the number of granulocytes and platelets at 24 h after MCA occlusion. Immunohistochemical analysis showed that tacrolimus reduced the number of blood vessels positively stained for ICAM-1, E-selectin and P-selection. These results suggested that tacrolimus limited attachment of granulocytes and platelets to blood vessels by inhibiting the expression of adhesion molecules and protected neuronal tissue from hypoxic insults.

Topics: Animals; Blood Platelets; Brain Ischemia; Cell Adhesion; Cerebral Cortex; Granulocytes; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Male; Microtubule-Associated Proteins; Neuroprotective Agents; Nitroimidazoles; Rats; Rats, Wistar; Tacrolimus

The immunosuppressant drug FK506 was found to be a potent neuroprotective agent in animal models of brain ischemia. However, the mechanisms underlying the action remain to be elucidated. The delayed rectifier K(+) channel has been implicated in ischemic injury and neuronal death in the brain. The aim of the present study is to investigate whether the neuroprotective action of FK506 results from blocking the K(+) channel. In acutely dissociated CA1 pyramidal neurons of rat hippocampus, superfusion of FK506 (0.01-100 microM) selectively inhibited the delayed rectifier K(+) current (I(K)) with an IC(50) value of 13.2+/-4.9 microM. The inhibition of I(K) by FK506 (10 microM) had a rapid onset, and then gradually reached a steady-state level. The inhibition was voltage-dependent, became more potent when the currents were elicited by strong depolarization. Moreover, FK506 (10 microM) caused marked negative shifts of the steady-state activation and inactivation curves of I(K), and accelerated its recovery from inactivation. Intracellular dialysis of FK506 (30 microM) was ineffective. The inhibition of I(K) by FK506 (10 microM) persisted under the low-Ca(2+) conditions that blocked the basal activity of protein phosphatase 2B (calcineurin). Rapamycin did not antagonize FK506 but mimicked it. Cyclosporin A inhibited I(K) only at 30 and 100 microM. Taken together, the results suggest that FK506 exert a direct inhibition on the delayed rectifier K(+) channel without involvement of calcineurin.

Topics: Animals; Animals, Newborn; Brain Ischemia; Calcineurin; Calcineurin Inhibitors; Dose-Response Relationship, Drug; Hippocampus; Immunosuppressive Agents; Ion Channel Gating; Membrane Potentials; Neural Inhibition; Neurons; Neuroprotective Agents; Organ Culture Techniques; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Tacrolimus

We recently demonstrated that a constitutively active form of calcineurin (CaN) is generated by calpain-mediated limited proteolysis following brain ischemia. The calpain-induced CaN activation mediated delayed neuronal death through translocation of nuclear factor of activated T-cells (NFAT) into nuclei after brain ischemia. We also previously demonstrated that activation of forkhead in rhabdomyosarcoma (FKHR), a forkhead transcription factor and substrate of protein kinase-B (Akt), mediated ischemia-induced neuronal death through Fas-ligand expression in gerbil hippocampus. FKHR activation occurred through decreased Akt activity and concomitant dephosphorylation mediated by undefined phosphatases. In this study, we show that phosphorylated Ser-256 of FKHR is dephosphorylated by constitutively active CaN and that in turn FKHR forms a complex with CaN that is translocated into nuclei after brain ischemia. After nuclear translocation of NFAT and FKHR, both NFAT and FKHR stimulated expression of Fas-ligand by binding to its promoter region. Consistent with activation of the Fas-ligand promoter by FKHR dephosphorylation, Fas-ligand expression increased 2 days after ischemia/reperfusion, and treatment with the CaN inhibitor FK506 inhibited that expression. These results suggest that FKHR is a downstream target of CaN and that constitutively active CaN mediates delayed neuronal death through Fas-ligand expression via up-regulation of both NFAT and FKHR transcriptional activity in brain ischemia.

Topics: Analysis of Variance; Animals; Brain Ischemia; Calcineurin; Cell Death; Electrophoretic Mobility Shift Assay; Enzyme Activation; Fas Ligand Protein; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation; Immunoprecipitation; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; NFATC Transcription Factors; Oncogene Protein v-akt; Phosphorylation; Protein Transport; Serine; Tacrolimus

The immunosuppressive agent FK506 (tacrolimus) has neuroprotective properties in an experimental model of cerebral ischemia. To improve the accuracy of clinical studies in acute stroke, a clinical dose setting should be based on the brain concentration, but not on the blood concentration of agents in humans. We have already established a measurement method using PET for FK506 concentration in the normal monkey brain, which could be applicable for human study; however, under ischemic conditions, in this study, we aimed to examine the brain concentration of FK506 in a monkey model of stroke.. Studies were performed on six male cynomolgus monkeys (Macaca fascicularis) and a middle cerebral artery (MCA) occlusion model was used. Regional cerebral blood flow (rCBF) was measured by an intravenous injection of [(15)O]H(2)O 165 min after MCA occlusion. FK506 (0.1 mg/kg) containing [(11)C]FK506 was intravenously injected into the monkeys 180 min after MCA occlusion, and dynamic PET images were acquired for 30 min after administration. FK506 concentrations in the brain were calculated in moles per liter (M) units using the specific activity of injected FK506.. MCA occlusion produced ischemia, confirmed by rCBF measurement before the administration of [(11)C]FK506. Fifteen minutes after FK506 (0.1 mg/kg) administration, the concentrations in the contralateral and ipsilateral cortex were 22.4+/-6.4 and 19.7+/-4.0 ng/g, respectively.. We successfully measured the brain concentration of FK506 in a monkey model of stroke. The difference between the contralateral and ipsilateral concentrations of FK506 was not significant. This characteristic that FK506 readily penetrates ischemic tissue as well as normal tissue might explain the neuroprotective effect of FK506 in the ischemic brain and is suitable for the treatment of stroke patients.

Topics: Animals; Brain Ischemia; Functional Laterality; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Injections, Intravenous; Macaca fascicularis; Male; Positron-Emission Tomography; Radiopharmaceuticals; Stroke; Tacrolimus

The blood-brain barrier possesses active transporters carrying brain-permeable xenobiotics back into the blood against concentration gradients. We demonstrate that multidrug resistance transporter (Mdr)-1 is upregulated on capillary endothelium after focal cerebral ischemia; moreover, Mdr-1 deactivation by pharmacological inhibition or genetic knockout preferably enhances the accumulation and efficacy of two neuroprotectants known as Mdr-1 substrates in the ischemic brain. We predict that Mdr-1 inhibition may greatly facilitate neuroprotective therapies.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blood-Brain Barrier; Brain; Brain Ischemia; Capillaries; Cerebrovascular Circulation; Endothelium, Vascular; Enzyme Inhibitors; Immunosuppressive Agents; Mice; Mice, Knockout; Neuroprotective Agents; Quinolines; Rifampin; Tacrolimus

In the present study, we investigated whether the protective effect of FK506 and cyclosporin A (CsA) against in vitro ischemic injury of astrocytes might be mediated through attenuation of cytosolic isoform of phospholipase A(2) (cPLA(2)) expression and activity as well as inhibition of arachidonic acid (AA) release. On the 21st day in vitro, cultures of rat astrocytes were subjected to ischemia-simulating conditions (combined oxygen glucose deprivation) for 8 h and exposed to FK506 (10 - 1,000 nM) and CsA (0.25 - 10 microM). Obtained data suggest the cross-talk between the action of 0.25 - 10 microM CsA as well as 1 microM FK506 on calcineurin (CaN) and cPLA(2) in anti-apoptotic signal transduction pathways. Moreover, we have shown that immunosuppressants at these concentrations protected glial cells against ischemia-induced apoptosis through the increase of cell viability, mitochondrial function restoration, and attenuation of oxidative stress. Finally, in our study, low concentrations of FK506 (10 and 100 nM) exerted limited effects on the assessed parameters. Our findings document a key role either for CaN or cPLA(2) expression attenuation and AA release inhibition in the antiapoptotic effect of FK506 and CsA in ischemic astrocytes.

Topics: Animals; Animals, Newborn; Antioxidants; Apoptosis; Arachidonic Acid; Astrocytes; Blotting, Western; Brain Ischemia; Calcineurin; Cell Survival; Cells, Cultured; Cyclosporine; Cytosol; Immunophilins; Immunosuppressive Agents; Membrane Potentials; Oxidative Stress; Phospholipases A; Phospholipases A2; Rats; Rats, Wistar; Receptor Cross-Talk; Signal Transduction; Tacrolimus

Ginkgo biloba exerts many pharmacological actions. It possesses antioxidant properties, the ability of neurotransmitter/receptor modulation and antiplatelet activation factor. This research is designed to investigate the neuroprotective effects of long-term treatment with EGb761 (a standard form of the extract of Ginkgo biloba leaf) in combination with MgSO(4), FK506, or MK-801 on the infarct volume of male gerbils' brain induced by unilateral middle cerebral artery occlusion (MCAO). Thirty-five gerbils fed a standard diet were intragastrically given water or EGb761 (100 mg/kg/day) for one week. Five randomized groups were established: control (n = 7), EGb761 (n = 8), EGb761 + MgSO(4) (n = 7), EGb761 + FK506 (n = 7), and EGb761 + MK-801 (n = 6). The three drug-combination groups were injected with MgSO(4) (90 mg/kg), FK506 (0.5 mg/kg), or MK-801 (1 mg/kg), respectively 30 min before MCAO. Gerbils were anesthetized and craniectomized to expose the right middle cerebral artery (MCA). The right MCA was constricted with an 8-0 suture to produce a permanent ligation for 24 hours. Postmortem infarct volumes were determined by quantitative image analysis of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections. Results showed that the total infarct volumes of the four treated groups either EGb761 alone or in combination with drugs were lower than the control group by 36.1% (EGb761 alone), 40.3% (EGb761 + MgSO(4)), 35.3% (EGb761 + FK506), and 56.4% (EGb761 + MK-801), respectively (p < 0.01). The main affected areas of the brain in the four treated groups were significantly focused between 4 and 6 mm from the frontal pole, when compared to the control group (p < 0.01). All animals in the five groups had infarctions in both cortex and subcortex. These results indicate that long-term pre-treatment of EGb761 administered either alone or in combination with drugs significantly effective neuroprotection on infarct volume in gerbil ischemic brains.

Topics: Animals; Brain Infarction; Brain Ischemia; Dizocilpine Maleate; Drug Therapy, Combination; Gerbillinae; Ginkgo biloba; Infarction, Middle Cerebral Artery; Magnesium Sulfate; Male; Neuroprotective Agents; Plant Extracts; Random Allocation; Tacrolimus; Treatment Outcome

The aim of the present study was to test the hypothesis that exposure of astrocytes depleted of glutathione (GSH) to simulated ischemia conditions in vitro and treated with immunosuppressant immunophilin ligands (cyclosporin A (CsA) and FK506) can increase intracellular GSH levels and that such mechanism may be responsible, at least in part, for their protective effects. In addition, we also compared the antioxidant properties of these immunosuppressants with N-acetylcysteine (NAC), a precursor of GSH synthesis. GSH depletion was induced by 24 h pretreatment with L-buthionine sulfoximine (BSO). Cultures of rat astrocytes were exposed to CsA (1-50 microM) and FK506 (1-1000 nM) and NAC (100 or 200 microM). We examined the effects of these compounds on apoptosis, cell viability, reactive oxygen species production and GSH content. Our study demonstrated that toxicity of simulated ischemia conditions were enhanced when intracellular GSH was depleted, and immunosuppressants (especially 100 nM FK506 and 10 microM CsA) effectively prevented ischemia toxicity in GSH depleted astrocytes. In addition, we have shown that interfering with the generation of GSH and attenuation, the rise of oxidative stress level by NAC may be a powerful tool for prevention of ischemia-induced glial cell damage.

Topics: Acetylcysteine; Animals; Apoptosis; Astrocytes; Benzimidazoles; Brain Ischemia; Cell Separation; Cells, Cultured; Cyclosporine; Fluorescent Dyes; Free Radical Scavengers; Glutathione; Immunophilins; Immunosuppressive Agents; Ligands; Mitochondria; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Tacrolimus; Tetrazolium Salts; Thiazoles

Death-associated protein kinase (DAPK) is a calcium calmodulin-regulated serine/threonine protein kinase involved in ischemic neuronal death. In situ hybridization experiments show that DAPK mRNA expression is up-regulated in brain following a global ischemic insult and down-regulated in ischemic tissues after focal ischemia. DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo. A similar dephosphorylation pattern is detected in primary cortical neurons subjected to oxygen glucose deprivation or N-methyl-D-aspartate (NMDA)-induced toxicity. Both a calcineurin inhibitor, FK506, and a selective NMDA receptor antagonist, MK-801, inhibit the dephosphorylation of DAPK after in vitro ischemia. This indicates that DAPK could be activated by NMDA receptor-mediated calcium flux, activation of calcineurin, and subsequent DAPK dephosphorylation. Moreover, concomitantly to dephosphorylation, DAPK is proteolytically processed by cathepsin after ischemia. Furthermore, a selective DAPK inhibitor is neuroprotective in both in vitro and in vivo ischemic models. These results indicate that DAPK plays a key role in mediating ischemic neuronal injury.

Topics: Animals; Apoptosis Regulatory Proteins; Brain Ischemia; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Death-Associated Protein Kinases; Dizocilpine Maleate; Enzyme Activation; Enzyme Inhibitors; Glucose; Male; Oxygen; Phosphorylation; Rats; Rats, Wistar; RNA, Messenger; Tacrolimus

The efflux of mitochondrial protein cytochrome C to cytoplasm is one of the key events of mitochondrial dysfunction observed in post-ischemic pathology. We investigated the effect of intra-carotid infusion of 5-10 mg/kg of cyclosporin A (CsA) on the neuronal survival in CA1 sector of hippocampus and on the subcellular localization of cytochrome C in the model of 5 min gerbil brain ischemia. To discriminate between the immunosuppressive and the mitochondria protecting component of CsA action, we compared the effect of CsA with one other immunosuppressant FK506. Almost 75% of neurons in ischemia-affected brain area were saved after CsA but not after FK506 treatment. This protective effect was only observed when the drug was infused immediately upon reperfusion. Early CsA treatment was able to block an initial phase of cytochrome C release, occurring transiently at 30 min post-ischemia, an effect never observed after FK506 administration. We assessed the neuroprotective potency of CsA vs. FK506 in rat cortical primary culture treated with compounds that mimic destructive signals induced by brain ischemia. In all cases, neuronal death and cytochrome C release were evidently suppressed by CsA applied not later than 30 min after the initial insult. Thus, early treatment with CsA in vitro and after bolus intra-carotid injection in vivo can save neurons by inhibition of cytochrome C efflux to cytoplasm.

Topics: Analysis of Variance; Animals; Biological Transport; Blotting, Western; Brain Ischemia; Cell Count; Cell Death; Cerebral Cortex; Cyclosporine; Cytochromes c; Cytoplasm; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Gerbillinae; Glutamic Acid; Hippocampus; Hydrogen Peroxide; Immunosuppressive Agents; In Vitro Techniques; Ischemic Attack, Transient; Male; Microscopy, Confocal; Rats; Rats, Wistar; Staurosporine; Tacrolimus; Time Factors; Tubulin

The behavioral and neurohistological protective effects of tacrolimus (FK506) were examined in rats subjected to 15-min global forebrain ischemia. Learning and memory performance were evaluated in an aversive, non-food-motivated, eight-arm radial maze. In one experiment, naive rats were rendered ischemic, and 15 days later they were tested for acquisition of a spatial task (postoperative training). In a complementary experiment, rats were trained for 8 days and then subjected to ischemia (preoperative training); 15 days later (on Day 24 of testing) they were retested for retention of cognition. FK506 (1.0 mg/kg) was given intravenously at the beginning of reperfusion, followed by doses applied intraperitoneally 6, 24, 48 and 72 h postischemia. Behavioral performance was expressed by latency to find the goal box, and number of errors. Ischemia did not affect acquisition performance. In contrast, retention of cognition was markedly impaired by ischemia, particularly working memory (P<.05-.001). This ischemia-induced, retrograde amnesia was significantly reduced by FK506 compared to vehicle alone on Day 24, as measured by latency and working memory errors (P<.025). A neuroprotective effect of FK506 was also seen on working memory, when postischemic performance was compared to that prior to ischemia (P>.05, Day 24 vs. Day 8, paired samples), in contrast to the significant, retrograde amnesia found in the ischemic, vehicle-treated group (P<.01). FK506 also significantly reduced the extent of hippocampal CA1 cell loss; however, this effect did not correlate with behavior. The present results suggest that the histological, neuroprotective effect of FK506 may be accompanied by a reduction in cognitive impairment, as assessed in a novel, non-food-motivated, eight-arm radial maze after transient, global, cerebral ischemia in rats.

Topics: Animals; Brain Ischemia; Hypoxia, Brain; Male; Memory Disorders; Pyramidal Cells; Rats; Rats, Wistar; Tacrolimus

The mechanism of the neuroprotective effect of FK506 in relation to nitric oxide (NO) production has not been clarified in vivo. We have investigated the effect of FK506 on ischemia-induced NO production in association with the pathogenesis of delayed neuronal death (DND) in rats.. In vivo microdialysis was performed in the hippocampus of male Sprague-Dawley rats (250-350 g). Dialysate samples were collected every 3 min. In the ischemia group (n=16), global ischemia was induced for 21 min and reperfusion was achieved. In the FK506 treatment group (n=25), FK506 (1 mg/kg, i.v.) was administered 21 min prior to the onset of global ischemia. Sham operations were done (n=15). The levels of NO(2)(-) in the dialysate samples were determined by the Griess reaction. The animals were decapitated 7 days after ischemia. Coronal brain sections were stained with hematoxylin and eosin.. In the ischemia group, the NO(2)(-) level significantly increased during ischemia. In the FK506 treatment group, there was no significant change in the NO(2)(-) level during ischemia. In histological examinations, FK506 treatment showed a neuroprotective effect against DND.. The effect of FK506 inhibiting NO production contributes to the neuro-protective effect of FK506 on DND in the hippocampus.

Topics: Analysis of Variance; Animals; Brain Ischemia; Cell Count; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Hippocampus; Humans; L-Lactate Dehydrogenase; Male; Microdialysis; Neuroblastoma; Neurons; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Rats; Rats, Sprague-Dawley; Reperfusion; Staining and Labeling; Tacrolimus; Tetrazolium Salts; Thiazoles

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemic cell death with respect to cytochrome c translocation and DNA fragmentation, which are pivotal events in the necrotic and apoptotic signaling pathway, using permanent focal cerebral ischemia in rats. Immunohistochemically, cytochrome c was observed in the cytoplasm as early as 1 h after middle cerebral artery (MCA) occlusion in the infarcted hemisphere. Cytosolic release of cytochrome c after MCA occlusion was also confirmed by Western blot analysis and enzyme immunoassay. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) showed DNA fragmentation evolving in the ipsilateral cortex and the caudate putamen after 3 and 6 h, respectively, following MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, significantly attenuated the release of cytochrome c in the ischemic region, the number of TUNEL-positive cells in the ischemic penumbra zone, and the size of cortical ischemic lesions. This study demonstrated that tacrolimus ameliorated the accumulation of cytochrome c in the cytosol and the increase of TUNEL-positive cells induced by cerebral ischemia, indicating that the neuroprotective action of tacrolimus on ischemic brain injury caused by permanent focal cerebral ischemia could partially be attributed to the attenuation of the activation of the apoptotic execution machinery.

Topics: Animals; Brain Ischemia; Cerebral Infarction; Cytochromes c; DNA Fragmentation; Immunosuppressive Agents; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tacrolimus

Tacrolimus (FK506), a potent immunosuppressive drug, is effective in attenuating brain infarction after cerebral ischemia. However, there has been no report characterizing the neuroprotective action and therapeutic time window of tacrolimus systematically using different types of stroke models and extended observation periods. Therefore, we evaluated the neuroprotective effect of tacrolimus in three different animal models of cerebral ischemia: transient and permanent focal ischemia in rats and transient global ischemia in gerbils. Tacrolimus at doses higher than 0.1 mg/kg (i.v.) produced a statistically significant reduction in ischemic brain damage following permanent and transient focal ischemia in rats when administered immediately after the onset of ischemia. Tacrolimus (1 mg/kg, i.v.) demonstrated similar neuroprotective activity even after delayed administration (2 h after permanent or 1 h after transient focal ischemia). The neuroprotective effect of tacrolimus was still present 2 weeks after transient focal ischemia and 1 week after permanent focal ischemia. After transient global ischemia in gerbils, tacrolimus (1 mg/kg, i.v.) given immediately after reperfusion also produced long-lasting neuroprotective effects with a protective time-window of 1-2 h. Taken together, the results clearly indicate that tacrolimus exerts potent, long-term neuroprotective effects with a favorable therapeutic time-window, regardless of the model of cerebral ischemia. These results strengthen the notion that tacrolimus might be of clinical value for the treatment of acute stroke.

Topics: Animals; Brain Ischemia; Cell Count; Dose-Response Relationship, Drug; Male; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tacrolimus; Time Factors

The neuroprotective effect of the immunosuppressant agent FK506 was evaluated in rats after brain ischemia induced for 15 min in the 4-vessel occlusion model. In the first experimental series, single doses of 1.0, 3.0 or 6.0 mg FK506/kg were given intravenously (iv) immediately after ischemia. In the second series, FK506 (1.0 mg/kg) was given iv at the beginning of reperfusion, followed by doses applied intraperitoneally (ip) 6, 24, 48, and 72 h post-ischemia. The same protocol was used in the third series except that all 5 doses were given iv. Damage to the hippocampal field CA1 was assessed 7 or 30 days post-ischemia on three different stereotaxic planes along the septotemporal axis of the hippocampus. Ischemia caused marked neurodegeneration on all planes (P<0.001). FK506 failed to provide neuroprotection to CA1 both when applied iv as a single dose of 1.0, 3.0 or 6.0 mg/kg (experiment 1), and after five iv injections of 1.0 mg/kg (experiment 3). In contrast, the repeated administration of FK506 combining iv plus ip administration reduced CA1 cell death on all stereotaxic planes both 7 and 30 days post-ischemia (experiment 2; P<=0.01). Compared to vehicle alone, FK506 reduced rectal temperature in a dose-dependent manner (P<=0.05); however, this effect did not alter normothermia (37 C). FK506 reduced ischemic brain damage, an effect sustained over time and apparently dependent on repeated doses and on delivery route. The present data extend previous findings on the rat 4-vessel occlusion model, further supporting the possible use of FK506 in the treatment of ischemic brain damage.

Topics: Animals; Brain Ischemia; Cell Death; Dose-Response Relationship, Drug; Hippocampus; Ischemic Attack, Transient; Male; Neuroprotective Agents; Rats; Rats, Wistar; Tacrolimus; Time Factors

FK506 is an immunosuppressant also showing neuroprotection following cerebral ischemia. FK506 binds to intracellular proteins (FKBP) which have a wide range of functions but have in common the peptidyl-prolyl cis/trans isomerase activity. Following transient focal ischemia, we have analyzed the expression of FKBP12, 52 and 65 and the total FKBP enzyme activity. Furthermore, we have investigated the effect of FK506 on signal transduction in neurons and perfusion changes in the infarct area. After 90 min of transient middle cerebral artery occlusion in male rats the expression of FKBP12, 52 and 65 was analyzed by Western blot in FK506-treated and control animals and the peptidyl-prolyl cis/trans isomerase activity was determined. Magnetic resonance imaging was used to measure tissue perfusion, development of vasogenic edema and infarct size. To investigate the neuronal stress signal cascade, activating transcription factor 2 (ATF-2), Fas-ligand (Fas-L) and c-Jun expression and phosphorylation were analyzed by immunohistochemistry. FK506 decreased the cerebral infarct volume by 53% and reduced the cytotoxic edema. The total FKBP enzymatic activity in the infarct area was increased and blocked dose dependently by FK506. FKBP expression was selectively up-regulated by cerebral ischemia. FK506 treatment does not influence the expression patterns. c-Jun phosphorylation in neurons of the peri-infarct area and Fas-L expression was reduced by FK506 treatment whereas ATF-2 expression was preserved. Cerebral ischemic damage to the brain was reduced by FK506. It was shown for the first time that neuroprotection by FK506 also included the suppression of the cerebral peptidyl-prolyl cis/trans isomerase activity of FKBP in vivo whereas the expression levels of FKBP12, 52 and 65 following ischemia changed slightly and FK506 treatment does not suppress the expression patterns. However, changes of FKBP enzymatic activity result in suppression of the stress cell body response in the peri-infarct area as observed by suppression of c-Jun phosphorylation and Fas-L expression.

Topics: Activating Transcription Factors; Analysis of Variance; Animals; Blood Proteins; Blotting, Western; Brain Ischemia; Brain Mapping; Dose-Response Relationship, Drug; fas Receptor; Functional Laterality; Immunohistochemistry; Infarction, Middle Cerebral Artery; Magnetic Resonance Imaging; Male; Neurons; Neuroprotective Agents; Peptidylprolyl Isomerase; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Reperfusion; Tacrolimus; Tacrolimus Binding Proteins; Time Factors; Transcription Factors

Tacrolimus (FK506), an immunosuppressive drug, is known to have potent neuroprotective activity and attenuate cerebral infarction in experimental models of stroke. Here we assess the neuroprotective efficacy of tacrolimus in a nonhuman primate model of stroke, photochemically induced thrombotic occlusion of the middle cerebral artery (MCA) in cynomolgus monkeys. In the first experiment, tacrolimus (0.01, 0.032, or 0.1 mg/kg) was intravenously administered immediately after MCA occlusion, and neurologic deficits and cerebral infarction volumes were assessed 24 hours after the ischemic insult. Tacrolimus dose-dependently reduced neurologic deficits and infarction volume in the cerebral cortex, with statistically significant amelioration of neurologic deficits at 0.032 and 0.1 mg/kg and significant reduction of infarction at 0.1 mg/kg. In the second experiment, the long-term efficacy of tacrolimus on neurologic deficits and cerebral infarction was assessed. Vehicle-treated monkeys exhibited persistent and severe deficits in motor and sensory function for up to 28 days. A single intravenous bolus injection of tacrolimus (0.1 or 0.2 mg/kg) produced long-lasting amelioration of neurologic deficits and significant reduction of infarction volume. In conclusion, we have provided compelling evidence that a single dose of tacrolimus not only reduces brain infarction but also ameliorates long-term neurologic deficits in a nonhuman primate model of stroke, strengthening the view that tacrolimus might be beneficial in treating stroke patients.

Topics: Animals; Blood Pressure; Brain Ischemia; Cerebrovascular Circulation; Disease Models, Animal; Heart Rate; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Macaca fascicularis; Male; Neurologic Examination; Neuroprotective Agents; Recovery of Function; Tacrolimus

The search for an effective treatment for global ischemia following cardiac arrest has proved to be very difficult. However, studies by Uchino et al. show that the immunosuppressant cyclosporin A (CsA), when administered in such a way that the drug can bypass the blood brain barrier (BBB), dramatically reduces ischemic damage in rat forebrain preparations. An alternative immunosuppressant, FK506, is apparently less efficacious. Both CsA and FK506 are specific inhibitors of immunophilins, (CsA inhibits cyclophilins, FK506 inhibits FKBPs), and of calcineurin, a type 2B Ser/Thr phosphatase that is abundant in the central nervous system. The superiority of CsA may be partly attributable to its selective amelioration of mitochondrial damage, as assayed in vivo and in vitro. Our results suggest that pathways involving calcineurin and cyclophilins, particularly mitochondrial cyclophilin D, play pivotal roles in the development of ischemic brain damage. The present findings may inform the search for new drugs in the treatment of global ischemic damage to the brain, and in other organs.

Topics: Animals; bcl-Associated Death Protein; Brain; Brain Ischemia; Calcineurin; Calcineurin Inhibitors; Calcium; Carrier Proteins; Cyclophilins; Cyclosporine; Cytochromes c; Immunosuppressive Agents; Mitochondrial Swelling; Neuroprotective Agents; Peptidyl-Prolyl Isomerase F; Phosphorylation; Prosencephalon; Rats; Rats, Wistar; Tacrolimus

To explore effects of Immunosuppressant FK506 on signal transduction pathway. we studied changes in subcellular distribution of protein kinase Cgamma (PKCgamma), CaM kinase II (CaMKII), as well as changes of tyrosine phosphorylation levels after ischemia. Male Mongolian gerbils were divided into 3 groups; FK506 (1 mg/kg, 3 mg/kg) and vehicle. FK506 was administered intravenously after 5 min ischemia. At the designated time points (0 time, 5 min ischemia, 1 hour, or 24 hour recovery), heads were frozen and samples were taken from CAI subfield of hippocampus. Western blot analysis was carried out with specific antibodies for PKCgamma, CaMKII, and phosphotyrosine. FK506 administration significantly decreased translocation of PKCgamma and CaMKII at 24 h of recovery (p < 0.05, ANOVA followed by Student-Newman Keuls' test) in P2 fraction. The levels of tyrosine phosphorylated p160, p140, p100, p90, and p80 in P2 fraction were also significantly decreased with FK506 treatment at 24 h of recovery. The persistently elevated PKCgamma and CaMKII level in P2 fraction which may be related to cell death, are attenuated with FK506 treatment. FK506 may contribute to recover calcium homeostasis in the post ischemic phase and promote cell survival.

Topics: Animals; Brain Ischemia; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Gerbillinae; Hippocampus; Immunosuppressive Agents; Male; Phosphorylation; Phosphotyrosine; Protein Kinase C; Protein Kinases; Tacrolimus; Tissue Distribution; Tyrosine

Transient global or forebrain ischemia leads to severe brain damage following delayed neuronal cell death. We previously reported that cyclosporin A (CsA) provides near total suppression of brain damage in rat forebrain ischemia when allowed to pass the blood brain barrier, whereas Tacrolimus (FK506) is considerably less effective. We demonstrate herein that when administered prior to ischemic insult, both immunosuppressants equally block calcineurin, a type 2B Ser/Thr phosphatase, and efficiently inhibit dephosphorylation of pro-apoptotic protein Bad. CsA demonstrates more potent anti-ischemic effects than FK506, partially attributable to amelioration of mitochondrial damage as assayed in vivo and in vitro. These results suggest that pathways including calcineurin and cyclophilins, particularly mitochondrial cyclophilin D, play pivotal roles in ischemic brain damage. Since previous results have shown that CsA is efficacious also when administered after focal ischemia, the present findings give hints to clinical applications for new drugs for the treatment of ischemic damage in the brain as well as in the heart and liver.

Topics: Animals; Brain Ischemia; Calcineurin; Calcineurin Inhibitors; Cyclosporine; Ion Channels; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Neuroprotective Agents; Prosencephalon; Rats; Rats, Wistar; Tacrolimus

Although the macrolide immunosuppressant tacrolimus (FK506) is neuroprotective in animal models of focal and global cerebral ischaemia, the mechanism of this action is not known. FK506 inhibits the protein phosphatase calcineurin, whose substrates can include nitric oxide synthase (NOS), and the neuroprotective effect of FK506 has been attributed to inhibition of NOS activity. We have examined nitric oxide-mediated cyclic guanosine monophosphate (cGMP) accumulation in neonatal rat cerebellar prisms. As expected, N-methyl-D-aspartate (NMDA) induced a rapid, concentration dependent accumulation of cGMP that was inhibited by the NMDA receptor antagonist dizocilpine (MK801) and the NOS inhibitor L-nitro-arginine methyl ester. Phosphoserine immunopositivity following NMDA exposure was increased in the presence of FK506, confirming inhibition of calcineurin. However, FK506 had no effect on NMDA-stimulated cGMP accumulation. These findings suggest that the neuroprotective effect of FK506 may be mediated by mechanisms other than increased NOS phosphorylation.

Topics: Animals; Animals, Newborn; Brain Ischemia; Calcineurin; Calcineurin Inhibitors; Cerebellum; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Immunosuppressive Agents; Male; N-Methylaspartate; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phosphorylation; Phosphoserine; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tacrolimus

Mitochondrial respiratory activities and energy metabolism were measured in neonatal rat brains to evaluate the influence of transient intrauterine ischemia on the near-term fetus and to assess the effect of the immunosuppressant drug FK506 treatment. Transient intrauterine ischemia was induced by 30 min of right uterine artery occlusion at 17 days of gestation in Wistar rats. The vehicle or 1.0 mg/kg of FK506 was administered after 1 h of recirculation. All of the pups were delivered by cesarean section at 21 days of gestation and samples of cerebral cortical tissue were obtained from pups at 1 h after birth. The mitochondrial respiration was measured polarographically in homogenates. For the analysis of ATP, ADP, and AMP, neonatal brains were frozen in situ and fluorometric enzymatic techniques were used. In the neonatal cortical tissue exposed to ischemia, mitochondrial respiratory activities and ATP concentrations decreased significantly to approximately 59 and 67% of those in normoxic controls, respectively. The deterioration of both mitochondrial respiratory activities and high-energy phosphates was prevented by FK506, given 1 h after the start of recirculation. The present results indicate that transient intrauterine ischemia is accompanied by mitochondrial dysfunction and cellular bioenergetic failure in the neonatal rat brain and suggest that treatment with FK506 prevents the deterioration, even when administered after the ischemic periods.

Topics: Animals; Animals, Newborn; Brain; Brain Chemistry; Brain Ischemia; Cell Respiration; Energy Metabolism; Female; Fetal Hypoxia; Immunosuppressive Agents; Mitochondria; Oxygen Consumption; Pregnancy; Rats; Rats, Wistar; Tacrolimus

The potential neuroprotective effects of the novel nitro-derivate of aspirin (NCX4016) on permanent focal cerebral ischemia in spontaneously hypertensive rats (SHRs) was investigated. Reference compounds were acetylsalicilic acid (ASA) and FK506 (tacrolimus). Ten minutes after surgery, SHRs were randomly divided into four groups of ten, pharmacologically treated and sacrificed 24 h after treatment. Brains were removed and processed to measure infarct volume, 70 kDa heat shock protein (hsp70), glial fibrillary acidic protein (GFAP) and vimentin (Vim) immunoreactivity (IR), and apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. NCX-4016 significantly reduced total infarct volume compared to ASA (-20%, P < 0.05), FK506 (-18%, P < 0.05) and vehicle treatment (-20%, P < 0.05). Experimental groups did not differ in hsp70-IR and GFAP-IR. Conversely, hyperplastic astrocytes, measured by Vim-IR, were significantly lower in NCX-4016 than in the vehicle group (-36%, P<0.01). TUNEL assay indicated a significantly lower degree of apoptosis in NCX-4016 group than vehicle in both the homolateral (-27%, P < 0.01) and contralateral hemisphere (-29%, P < 0.05). These findings indicate that NO release associated with aspirin confers neuroprotective effects against ischemic injury.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Astrocytes; Brain; Brain Injuries; Brain Ischemia; Cell Survival; Functional Laterality; Glial Fibrillary Acidic Protein; HSP70 Heat-Shock Proteins; Immunohistochemistry; Immunosuppressive Agents; Male; Nerve Degeneration; Neurons; Neuroprotective Agents; Rats; Rats, Inbred SHR; Tacrolimus; Vimentin

The aim of this study was to monitor dynamic changes in energy-related metabolites in the cortex of gerbils subjected to cerebral ischemia by a dual probe microdialysis technique. Focal cerebral ischemia was produced in anesthetized gerbils by occlusion of the right common carotid artery and the right middle cerebral artery for 60 min. Two microdialysis probes were inserted into both sides of the cortex to simultaneously monitor extracellular glucose, lactate, pyruvate and glutamate. Dynamic and comparative changes in these analytes, on the ipsilateral and contralateral sides of the brain, were simultaneously monitored by liquid chromatography and a microdialysis analyzer. The present study demonstrated decreases in glucose and pyruvate, increases in lactate and glutamate on the ipsilateral side whereas all analytes remain constant on the contralateral side of cortex during cerebral ischemia. In vitro recovery of each microdialysis probe was performed to ensure the quality of experiments. The detection limits of pyruvate, glutamate, lactate and glucose were 0.2, 1.0, 2.0 and 20 microM, respectively. The intra- and inter-assay correlations were less than 5% in standard mixtures and pooled brain dialysates.

Topics: Animals; Brain Ischemia; Calibration; Cerebral Cortex; Extracellular Space; Gerbillinae; Glucose; Glutamic Acid; Lactates; Male; Microdialysis; Pyruvates; Tacrolimus

Topics: Animals; Brain Ischemia; Cerebral Cortex; Disease Models, Animal; Ischemic Attack, Transient; Male; Mice; Middle Cerebral Artery; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tacrolimus

FK506 (tacrolimus) is known as an inhibitor for calcineurin and is used in numerous research fields. It is not clear whether intravenously injected FK506 inhibits neuronal calcineurin. We measured the calcineurin activities of normal and postischemic rat hippocampi after intravenous injection of FK506 (3 mg/kg). Intravenously injected FK506 had no inhibitory effect on calcineurin activity in the hippocampi of normal and postischemic rats, whereas FK506 inhibited the calcineurin in vitro (purified enzyme, hippocampal homogenate, and hippocampal slice culture homogenate). Thus, it is considered that intravenously injected FK506 does not act on intraneuronal calcineurin and that several effects of FK506 are not due to the inhibition of neuronal calcineurin.

Topics: Animals; Brain Ischemia; Calcineurin Inhibitors; Culture Techniques; Hippocampus; Injections, Intravenous; Kinetics; Male; Rats; Rats, Wistar; Tacrolimus; Tissue Extracts

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

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

Protein phosphorylation and dephosphorylation mediated by protein kinases and protein phosphatases, respectively, represent essential steps in a variety of vital neuronal processes that could affect susceptibility to ischemic stroke. In this study, the role of the neuron-specific gamma isoform of protein kinase C (gammaPKC) in reversible focal ischemia was examined using mutant mice in which the gene for gammaPKC was knocked-out (gammaPKC-KO). A period of 150 minutes of unilateral middle cerebral artery and common carotid artery (MCA/CCA) occlusion followed by 21.5 hours of reperfusion resulted in significantly larger (P < 0.005) infarct volumes (n = 10; 31.1+/-4.2 mm3) in gammaPKC-KO than in wild-type (WT) animals (n = 12; 22.6+/-7.4 mm3). To control for possible differences related to genetic background, the authors analyzed Balb/cJ, C57BL/6J, and 129SVJ WT in the MCA/CCA model of focal ischemia. No significant differences in stroke volume were detected between these WT strains. Impaired substrate phosphorylation as a consequence of gammaPKC-KO might be corrected by inhibition of protein dephosphorylation. To test this possibility, gammaPKC-KO mice were treated with the protein phosphatase 2B (calcineurin) inhibitor, FK-506, before ischemia. FK-506 reduced (P < 0.008) the infarct volume in gammaPKC-KO mice (n = 7; 24.6+/-4.6 mm3), but at this dose in this model, had no effect on the infarct volume in WT mice (n = 7; 20.5+/-10.7 mm3). These results indicate that gammaPKC plays some neuroprotective role in reversible focal ischemia.

Topics: Animals; Brain; Brain Ischemia; Calcineurin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Female; Gene Expression Regulation, Enzymologic; Genetic Predisposition to Disease; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Isoenzymes; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Protein Kinase C; Tacrolimus

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.

Topics: Animals; Brain Ischemia; Cell Cycle; Ciclopirox; Dose-Response Relationship, Drug; Flavonoids; Growth Inhibitors; Male; Nerve Growth Factor; Neurites; PC12 Cells; Piperidines; Pyridones; Pyrrolidines; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus

Pyruvate dehydrogenase is one of the mitochondrial enzymes considered important in the regulation of oxidative metabolism. To further understand the relationship between its activity and ischemic brain damage we conducted three experiments. We studied the effects of (1) duration of cerebral ischemia, (2) the Ca2+ channel blocker, nicardipine, and (3) the immunosuppressant, FK506, on PDH activity and energy metabolites during ischemia and reperfusion. In the first study we also measured regional cerebral blood flow (rCBF). (1) Increasing the duration of the ischemic insult delayed the deactivation of PDH, slowed the resynthesis of high energy phosphates and the clearance of lactate, and impaired recovery of rCBF. Additionally, (2) nicardipine normalized PDH activities and improved the impaired metabolism after reperfusion, and (3) FK506 did not effect PDH activity, but significantly improved the impaired metabolism during the early phase of reperfusion. From these studies we conclude that PDH plays a role in the recovery of metabolism during reperfusion, and both nicardipine and FK506 improve metabolism during the early phase of reperfusion.

Topics: Animals; Brain; Brain Ischemia; Calcium Channel Blockers; Energy Metabolism; Gerbillinae; Immunosuppressive Agents; Nicardipine; Pyruvate Dehydrogenase Complex; Rats; Reperfusion; Tacrolimus; Time Factors

We investigated post-ischemic changes in FK506 binding protein (FKBP) in the brain after transient global ischemia in gerbils or transient focal ischemia in rats. [3H]FK506 was used to label FKBP as a immunophilin. In transient global ischemia, [3H]FK506 binding showed a transient reduction in the frontal cortex only 1 h after recirculation. In the striatum, the dorsolateral part exhibited a significant increase in [3H]FK506 binding 5, 24 and 48 h after ischemia. However, the ventromedial part showed a transient elevation in [3H]FK506 binding 24 h after ischemia. Thereafter, the ventromedial part showed no conspicuous change in [3H]FK506 binding up to 7 days after ischemia. The dorsolateral part also showed no significant change in [3H]FK506 binding 7 days after ischemia. In the hippocampus and thalamus, [3H]FK506 binding was unchanged in the stratum radiatum of the hippocampal CA1 sector, hippocampal CA3 sector, dentate gyrus and thalamus up to 7 days after ischemia. However, the stratum oriens of the hippocampal CA1 sector showed a significant reduction in [3H]FK506 binding 48 h and 7 days after ischemia. A histological study showed that transient cerebral ischemia caused a severe damage in the striatum and hippocampal CA1 sector. In a model of transient focal ischemia, a marked increase in [3H]FK506 binding was also found in the striatum and cerebral cortex where severe infarctions were observed. These results demonstrate that post-ischemic change in [3H]FK506 binding between the striatum and hippocampus may be produced by different mechanisms. Furthermore, our findings suggest that immunophilins may play some role in the pathogenesis of ischemic diseases.

Topics: Animals; Autoradiography; Brain; Brain Ischemia; Carrier Proteins; DNA-Binding Proteins; Gerbillinae; Heat-Shock Proteins; Immunosuppressive Agents; Male; Rats; Rats, Wistar; Tacrolimus; Tacrolimus Binding Proteins

Neuroprotective effects of immunosuppressive agents have been shown in cerebral ischemia. To investigate the role of immunosuppressive agents in chronic cerebral ischemia and to design a drug protocol with safe therapeutic windows, we examined the effects of FK506, a potent immunosuppressive agent, on chronic cerebral ischemia. Both common carotid arteries were ligated in 73 male Wistar rats. Fifty-eight of these rats received a chronic injection of FK506 (0.2, 0.5, 1.0 mg/kg) and the remaining 15 received a vehicle solution injection. Microglia/macrophage was investigated with immunohistochemistry for leukocyte common antigen and major histocompatibility complex, and astroglia was examined with glial fibrillary acidic protein as markers. White matter rarefaction and the number of immunopositive glial cells were assessed from 7 to 30 days after the ligation. In the vehicle-treated animals, there was persistent and extensive activation of the microglia/macrophages and astroglia in the white matter, including the optic nerve, optic tract, corpus callosum, internal capsule, anterior commissure and traversing fiber bundles of the caudoputamen. In the FK506-treated rats, the number of activated microglia/macrophages was significantly reduced in a dose-dependent manner (p<0.01) as compared to the vehicle-treated rats. Rarefaction of the white matter was also inhibited by FK506 in a dose-dependent manner (p<0. 01). Thus, a clinically-relevant dosage of FK506 attenuated both glial activation and white matter changes in chronic cerebral ischemia in the rat. These results indicate a potential use for FK506 in cerebrovascular diseases.

Topics: Animals; Blood Glucose; Blood Urea Nitrogen; Brain; Brain Ischemia; Chronic Disease; Dose-Response Relationship, Drug; Immunohistochemistry; Male; Neuroprotective Agents; Optic Nerve; Rats; Rats, Wistar; Tacrolimus; Time Factors; Transaminases

The purpose of the present study was to compare the characteristics of the photochemical-induced thrombotic occlusion model and the thermocoagulated occlusion model of the middle cerebral artery in rats. We evaluated the neuroprotective effects of a NMDA receptor antagonist, (+)-MK-801 (dizocilpine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine), an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, YM90K (6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride), a Ca2+ channel antagonist, S-312-d (S-(+)-methyl-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)-thieno[2 ,3-b]pyridine-5-carboxylate), the radical scavengers, MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) and EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyl-tridecyl)-2H-1-be nzopyran-6yl-hydrogen phosphate] potassium salt), and a calcineurin inhibitor, FK506 (tacrolimus, Prograf). Although all tested agents in the present study attenuated the brain damage in the photochemical-induced thrombotic occlusion model, the radical scavengers did not attenuate the brain damage in the thermocoagulated occlusion model. The time course of brain damage and brain edema formation in the two models was examined. The time course of brain damage was not different in the two models, but the time course of brain edema was quite different. Brain edema formation in the photochemical-induced thrombotic occlusion model was significantly greater (P < 0.01) than that in the thermocoagulated occlusion model at all time point studied until 24 h after occlusion of the middle cerebral artery. The present study suggests that the photochemical-induced thrombotic occlusion model has characteristics of both permanent ischemia and ischemia-reperfusion.

Topics: Animals; Brain Edema; Brain Ischemia; Disease Models, Animal; Dizocilpine Maleate; Electrocoagulation; Immunosuppressive Agents; Injections, Intraperitoneal; Injections, Intravenous; Intracranial Embolism and Thrombosis; Male; Neuroprotective Agents; Quinoxalines; Rats; Rats, Sprague-Dawley; Tacrolimus

We examined the effects of FK506, a specific inhibitor of calcineurin, on the binding capacity of cyclic AMP-dependent protein kinase (cAMP-DPK) in gerbils subjected to 2-h cerebral hemispheric ischemia. FK506 (0.1 mg/kg) was infused intravenously at 15 min prior to the induction of ischemia by common carotid artery occlusion. The binding capacity of cAMP-DPK was evaluated by autoradiographic analysis of the cAMP binding, and cerebral blood flow (CBF) was measured by the [14C] iodoantipyrine method. In the sham-operated gerbils. FK506 significantly increased mean arterial blood pressure and tended to decrease CBF, suggesting that FK506 may constrict systemic blood vessels as well as cerebral blood vessels. On the other hand, cAMP binding was not altered by FK506 in the sham-operated gerbils. In the ischemia group of gerbils, FK506 prevented any significant reduction of cAMP binding in the hippocampus CA1 and cerebral cortices on the ischemic side, whereas it exerted no significant influence on the cAMP binding of the nonischemic side. The values of CBF were comparable between the vehicle-treated gerbils and FK506-treated gerbils in the ischemic regions. Preservation of cAMP binding indicates that intracellular signal transduction via cAMP-DPK can be maintained by FK506 despite ischemia, suggesting that this agent may be beneficial for reducing ischemic tissue damage.

Topics: Animals; Autoradiography; Brain; Brain Ischemia; Calcineurin; Calmodulin-Binding Proteins; Cerebrovascular Circulation; Cyclic AMP-Dependent Protein Kinases; Female; Gerbillinae; Male; Phosphoprotein Phosphatases; Reference Values; Tacrolimus

The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, although in vitro studies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The present in vivo study demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56-58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%. The temporal window for FK506 neuroprotection was defined in studies demonstrating efficacy using intravenous administration at 120 min, but not 180 min, after middle cerebral artery occlusion. The noncompetitive NMDA receptor antagonist MK801 reduced both ischemic and excitotoxic damage. Histopathological data concerning striatal quinolinate lesions were replicated in neurochemical experiments. MK801, but not FK506, attenuated the loss of glutamate decarboxylase and choline acetyltransferase activity induced by intrastriatal injection of quinolinate. The contrasting efficacy of FK506 in ischemic and excitotoxic lesion models cannot be explained by drug pharmacokinetics, because brain FK506 content rose rapidly using both treatment protocols and was sustained at a neuroprotective level for 3 d. Although these data indicate that an antiexcitotoxic mechanism is unlikely to mediate the neuroprotective action of FK506 in focal cerebral ischemia, the finding that intravenous cyclosporin A (20 mg/kg) reduced ischemic cortical damage is consistent with the proposed role of calcineurin.

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Choline O-Acetyltransferase; Dizocilpine Maleate; Glutamate Decarboxylase; Injections, Intraperitoneal; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tacrolimus

The effect of the immunosuppressant FK506 on ischaemic neuronal damage was studied in a rat model of transient cerebral ischemia induced by occlusion of both common carotid arteries in combination with hypotension for 10 min. Neuronal damage was assessed morphologically after 4 days of recovery. Treatment with FK506, given at a dose of 2 mg kg-1 by intraperitoneal injections 30 min prior to ischemia and once daily during recovery, decreased neuronal damage by 52% in the hippocampal CA1 region and by 48% in the temporal cortex. The protection was not due to diminished body temperature or a marked reduction of ischaemia-induced synaptic overflow of glutamate. We propose that FK506 decreases neuronal damage either by inhibiting calcineurin-mediated events or by preserving mitochondrial function.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Glutamic Acid; Hippocampus; Male; Rats; Rats, Wistar; Tacrolimus

Tacrolimus (FK506) is a potent immunosuppressant that is presently in clinical use for prevention of allograft rejection. Recently, animal studies reporting significant reductions in the volume of tissue damage associated with cardiac, hepatic, and cerebral ischemia suggest that tacrolimus may also be of use in the clinical management of stroke. In the present study, we examine whether the neuroprotective effects of tacrolimus, as assessed by histological outcome, are accompanied by an amelioration of the skilled motor deficits induced in the rat by middle cerebral artery occlusion (MCAO).. Animals were trained to perform a skilled paw-reaching task before MCAO by perivascular microinjections of endothelin-1. Tacrolimus (1 mg/kg, n = 6) or vehicle (n = 6) was administered by intravenous infusion 1 minute after MCAO. After a 5-day postoperative recovery period, the rats were retested for skilled paw-reaching ability for an additional 9 days.. In vehicle-treated rats, MCAO resulted in a profound bilateral impairment in skilled paw use. Rats treated with tacrolimus, although still impaired, performed significantly better than those treated with vehicle alone (P < .01). Histological analysis, 14 days after occlusion, confirmed the neuroprotective efficacy of tacrolimus with a 66% reduction in the volume of hemispheric brain damage produced by MCAO (P < .01).. The present studies show that tacrolimus not only protects neural tissue from focal cerebral ischemia but also significantly ameliorates the deficits in skilled motor ability produced by this lesion. These data provide further support for the view that tacrolimus may be of use in the treatment of stroke.

Topics: Animals; Brain Ischemia; Cerebral Arteries; Constriction; Endothelin-1; Male; Movement Disorders; Neuroprotective Agents; Psychomotor Performance; Rats; Tacrolimus

The immunosuppressive action of the drug FK506 involves inhibition of calcineurin in T-lymphocytes by a complex of FK506 and an FK506 binding protein, FKBP12, a member of the immunophilin protein family. The functional role of brain immunophilins is, however, unclear. We show here that FK506 is a powerful neuroprotective agent in an in vivo model of focal cerebral ischaemia when administered up to 60 min post-occlusion. The minimum effective neuroprotective dose is comparable with the immunosuppressant dose in humans, suggesting that FK506 may have clinical potential for the treatment of stroke. Although the related immunosuppressants rapamycin and cyclosporin failed to reduce brain damage, the finding that rapamycin pretreatment blocked the effect of FK506 confirms a role for immunophilins in the neuroprotective mechanism.

Topics: Animals; Brain Ischemia; Carrier Proteins; Cerebral Cortex; Corpus Striatum; Cyclosporine; Heat-Shock Proteins; Humans; Immunosuppressive Agents; Male; Polyenes; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins