tacrolimus and Infarction--Middle-Cerebral-Artery

The development of a diagnostic technology that can accurately determine the pathological progression of ischemic stroke and evaluate the therapeutic effects of cerebroprotective agents has been desired. We previously developed a novel PET probe, 2-tert-butyl-4-chloro-5-{6-[2-(2-(18)F-fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ([(18)F]BCPP-EF) for detecting activity of mitochondrial complex I (MC-I). This probe was shown to visualize neuronal damage in the living brain of rodent and primate models of neurodegenerative diseases. In the present study, [(18)F]BCPP-EF was applied to evaluate the therapeutic effects of a neuroprotectant, liposomal FK506 (FK506-liposomes), on cerebral ischemia/reperfusion (I/R) injury in transient middle cerebral artery occlusion rats. The PET imaging using [(18)F]BCPP-EF showed a prominent reduction in the MC-I activity in the ischemic brain hemisphere. Treatment with FK506-liposomes remarkably increased the uptake of [(18)F]BCPP-EF in the ischemic side corresponding to the improvement of blood flow disorders and motor function deficits throughout the 7 days after I/R. Additionally, the PET scan could diagnose the extent of the brain damage accurately and showed the neuroprotective effect of FK506-liposomes at Day 7, at which 2, 3, 5-triphenyltetrazolium chloride staining couldn't visualize them. Our study demonstrated that the PET technology using [(18)F]BCPP-EF has a potent capacity to evaluate the therapeutic effect of drug candidates in living brain.

Topics: Animals; Cerebral Infarction; Electron Transport Complex I; Infarction, Middle Cerebral Artery; Liposomes; Male; Motor Activity; Neuroprotective Agents; Positron-Emission Tomography; Rats; Rats, Wistar; Tacrolimus

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

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

Stroke is the leading cause of disability in the industrialized world, and the development of pharmacologic strategies to promote poststroke recovery is of paramount importance. GM1485, a nonimmunosuppressive immunophilin ligand, promotes regeneration of multiple cell types following injury. In the present study, we evaluated the effect of GM1485 treatment on functional recovery and neurogenesis following rat stroke. Transient cerebral ischemia was induced in rats receiving daily GM1485 (5 mg/kg) beginning 24 hr postischemia and continuing for a total of 6 weeks. Neurological function was evaluated over this period using a battery of neurobehavioral tests, and immunostaining for stem-cell markers was performed following animal sacrifice. An in vitro model of oxidative stress was also employed to evaluate the ability of GM1485 to mediate stem-cell-like induction and plasticity. GM1485-treated rats demonstrated improved neurological function as well as increased Sox2(+) cells in the ipsilateral SVZ and striatum relative to vehicle-treated rats. Additionally, GM1485-treated fibroblasts subjected to oxidative stress were reprogrammed to a stem-cell-like phenotype and were able to differentiate down a neuronal lineage. These data demonstrate that GM1485 administration improves neurological function and is consistent with an upregulation of endogenous neurogenesis following stroke in rats. Further experiments are necessary to characterize the molecular pathways involved in these processes.

Topics: Animals; Disease Models, Animal; Immunophilins; Infarction, Middle Cerebral Artery; Infusions, Intravenous; Ligands; Male; Nerve Growth Factors; Nerve Regeneration; Primary Cell Culture; Rats; Rats, Wistar; Recovery of Function; Tacrolimus; Treatment Outcome

The immunosuppressant FK506 (tacrolimus) is neuroprotective in experimental models of cerebral ischemia. However, the precise mechanisms underlying this neuroprotection remain unknown. In the present study, we hypothesized that FK506 treatment could protect rat brain from oxidative injuries through antioxidative and anti-inflammatory pathways after ischemia-reperfusion injury.. Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 120 minutes, followed by reperfusion. Animals received a single injection of FK506 (0·3 mg/kg) or vehicle intravenously at 30 minutes after ischemic induction. Infarct volume and neurological performance were evaluated at 24 hours after reperfusion. Immunohistochemical analysis for 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-deoxyguanosine (8-OHdG), ionized calcium-binding adapter molecule 1 (Iba-1), and tumor necrosis factor-alpha (TNF-alpha) were conducted at 24 hours after reperfusion.. FK506 significantly reduced infarct volume (61·7%; P=0·01) and improved neurological deficit scores (P<0·05) 24 hours after reperfusion compared to vehicle. In FK506-treated rats, accumulation of 4-HNE (P<0·01) and 8-OHdG (P<0·01) was significantly suppressed in the cerebral cortex 24 hours after reperfusion. In addition, FK506 markedly reduced microglial activation (P<0·01) and TNF-alpha expression (P<0·01).. These results demonstrate that FK506 may have antioxidant as well as anti-inflammatory effects and reduces ischemic damage following cerebral infarction.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Antioxidants; Brain; Calcium-Binding Proteins; Cerebral Infarction; Cerebrovascular Circulation; Deoxyguanosine; Disease Models, Animal; Humans; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Magnetic Resonance Imaging; Male; Microfilament Proteins; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Sprague-Dawley; Tacrolimus; Tumor Necrosis Factor-alpha

Although, astrocytes are more resistant than neurons to ischemic injury, astrocyte death has been demonstrated in animal models of brain ischemia. Astrocytes death after ischemia/reperfusion may strongly affect neuronal survival because of the absence of their trophic and metabolic support to neurons, and astrocytic glutamate uptake. Early signals involved in astrocytes death are poorly understood. We demonstrated enhanced and mostly cytoplasmic activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) during glutamate-induced apoptosis of cultured astrocytes. Treatment with UO126, inhibitor of MEK1, threo-beta-benzyloxyaspartic acid, glutamate transporter inhibitor, and FK506, a cytoprotective drug prevented ERK activation and glutamate-induced apoptosis. Over-expression of ERK dual specificity phosphatases 5 and 6 reduced apoptosis in transfected astrocytes. Prolonged ERK1/2 activation was observed in ischemic brain: in the nucleus and cytoplasm of astrocytes in the cerebral cortex, and exclusively in the cytoplasm of astrocytes in the striatum. Global gene expression profiling in the cortex revealed that FK506 blocks middle cerebral artery occlusion-induced expression of numerous genes associated with ERK signaling pathway and apoptosis. The results demonstrate a pro-apoptotic role of sustained activation of ERK1/2 signaling in glutamate-induced death of astrocytes and the ability of FK506 to block both ERK activation and astrocytic cell death in vitro and in ischemic brains.

Topics: Amino Acid Transport System X-AG; Animals; Animals, Newborn; Apoptosis; Astrocytes; Butadienes; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Glutamic Acid; Hypoxia-Ischemia, Brain; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Male; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Neuroprotective Agents; Nitriles; Rats; Rats, Wistar; Tacrolimus

The aim of this study was to evaluate the effect of tacrolimus on recombinant tissue-plasminogen activator (rt-PA)-induced hemorrhagic transformation, and to characterize its suppressive action for hemorrhage. Thrombotic occlusion of the middle cerebral artery (MCA) was induced by photochemical reaction in spontaneously hypertensive rats, and hemorrhagic scores and brain damage were measured 24 h after MCA occlusion. Administration of rt-PA 3 h after MCA occlusion significantly worsened spontaneous hemorrhagic changes and tended to aggravate brain damage. Hematoma was observed in 7 of 15 rats treated with rt-PA, and 0 of 15 rats in the control group. Tacrolimus alone administered intravenously 3 h after MCA occlusion did not produce any hemorrhagic changes. The combined treatment of tacrolimus followed by rt-PA significantly decreased the incidence of hematoma and brain damage in comparison with that of the rt-PA treated group. Permeability of the blood-brain-barrier (BBB) detected by extravasations of Evans blue was investigated 6 h after MCA occlusion, as was the integrity of microvascular endothelial cells as determined by immunohistochemical assessment of the prevalence of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). Combined treatment of rt-PA with tacrolimus reduced the rt-PA-induced extravasation of Evans blue and preserved CD31-positive cells in the ischemic hemisphere. Thus, tacrolimus was able to reduce the rt-PA-induced hemorrhagic transformation, which might be due to the protective effects on cerebral microvascular endothelial cells after thrombotic cerebral ischemia during the acute phase of cerebral ischemia. In conclusion, the combination of rt-PA with tacrolimus may be useful for decreasing the risk of thrombolytic therapy.

Topics: Animals; Blood-Brain Barrier; Brain; Endothelial Cells; Evans Blue; Immunohistochemistry; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Male; Microvessels; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Inbred SHR; Recombinant Proteins; Tacrolimus; Tissue Plasminogen Activator

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 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

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

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

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

It has been reported that immunosuppressant FK506 inhibited ischemic neuronal injury in forebrain ischemia or transient focal cerebral ischemia, but the mechanisms of the neuroprotective effect have not been clarified. In permanent focal cerebral ischemia, we investigated whether FK506 caused remission of brain infarction, and how mechanism was concerned. Male Balb/c mice were subjected to permanent middle cerebral artery (MCA) occlusion. They were treated with 1.0 or 3.0 mg/kg FK506 or vehicle 30 min before ischemia. Infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) method after 24 h. Cytochrome c release from mitochondria was evaluated by Western blotting and immunocytochemistry after ischemia. Simultaneously, the immunoreactivity of total and phosphorylated BAD was also studied using immunocytochemistry. We demonstrated that pretreatment with 3.0 mg/kg FK506 salvaged the tissue damage in the infarct rim and significantly reduced infarct volume to 75.5% (P<0.05), and FK506 inhibited cytochrome c release on 6 h after ischemia for Western blot analysis (P<0.05). Immunocytochemical study showed that permanent MCA occlusion increased the amount of cytochrome c and total BAD in the cytosol, but not phosphorylated BAD, in the ischemic core and the infarct rim as early as 1 h after ischemia, and FK506 inhibited the increases in the infarct rim. The results suggest that FK506 may, at least in part, ameliorate tissue damage due to permanent focal cerebral ischemia in the infarct rim through maintaining BAD turnover and inhibiting cytochrome c release from mitochondria.

Topics: Analysis of Variance; Animals; bcl-Associated Death Protein; Blotting, Western; Brain Infarction; Carrier Proteins; Cytochromes c; Dose-Response Relationship, Drug; Immunohistochemistry; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred BALB C; Neocortex; Phosphorylation; Tacrolimus; Tetrazolium Salts; Time Factors

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

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 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

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

Cyclosporin A (CsA) reduces ischemic brain damage when administered in such a way that its penetration across the blood-brain barrier is enhanced. Since only pretreatment has previously been used in focal ischemia, the objective of the present study was to establish whether posttreatment is efficacious and to assess the window of therapeutic opportunity for CsA. To that end, CsA was given 5 min to 6 h after the start of reperfusion following 2 h transient ischemia, and infarct volume was assessed after 48 h by triphenyltetrazolium chloride staining. Attempts were made to circumvent the BBB to CsA by an intracerebral needle lesion, by an increase in the intravenous CsA dose, or by osmotic opening with intracarotid mannitol. The results were compared to those obtained with FK506. Intravenous CsA in a dose of 10 mg/kg failed to reduce infarct volume, unless preceded by a needle lesion. That procedure, and an increase in CsA dose to 50 mg/kg, reduced infarct volume to about 50% of control, but the higher dose had toxic side effects. The coupled intracarotid infusion of mannitol and CsA (10 mg/kg) was more efficacious, without overt side effects. However, mannitol proved dispensable since CsA alone reduced infarct volume to 30% of control, with a therapeutic window of 3-6 h. When given after 5 min of reflow, CsA reduced infarct volume to 10% of control and was clearly more neuroprotective than FK506. Possibly, this is because CsA blocks the mitochondrial permeability transition pore which is opened under adverse conditions.

Topics: Animals; Blood Glucose; Blood-Brain Barrier; Body Temperature; Brain Injuries; Carbon Dioxide; Carotid Arteries; Cyclosporine; Diuretics, Osmotic; Hydrogen-Ion Concentration; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Injections, Intra-Arterial; Ischemic Attack, Transient; Male; Mannitol; Osmosis; Oxygen; Rats; Rats, Wistar; Sodium Chloride; Tacrolimus; Wounds, Stab