tacrolimus and Ischemic-Attack--Transient

Treatment with FK506, an inhibitor of Ca2+/calmodulin dependent phosphatase (calcineurin, CaN), within 1 hr after transient ischemia afforded protection from apoptotic death in CA1 pyramidal neurons. To investigate isoform-specific roles of CaN in the neuronal cell death, we localized CaN A alpha and CaN A beta in the gerbil hippocampus using isoform-specific antibodies. In control gerbils, immunoreactions of both isoforms were highly enriched in hippocampal CA1 pyramidal neurons. Four to seven days after the induced ischemia, immunoreactivities of both isoforms were markedly reduced in the CA1 pyramidal cell and lacunosum-molecular layers. The CaN A alpha immunoreactivity was increased in the CA1 radiatum and oriens layers, whereas that of CaN A beta was enhanced in reactive astrocytes in the CA1 region. These findings suggest that CaN A alpha is involved in sprouting of afferent fibers in CA1 and that CaN A beta is involved in the reaction of astrocytes such as assembly of glial fibril acidic protein.

Topics: Animals; Apoptosis; Calcineurin; Calcineurin Inhibitors; Disease Models, Animal; Gerbillinae; Glial Fibrillary Acidic Protein; Hippocampus; Immunosuppressive Agents; Ischemic Attack, Transient; Isoenzymes; Neurons; Tacrolimus

Recirculation following 2 h of focal ischemia due to transient middle cerebral artery (MCA) occlusion has previously been found to be accompanied by an initial, partial recovery of the cellular bioenergetic state and of mitochondrial respiratory functions, with secondary deterioration during the first 2-4 h of reflow. Both the free radical spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) and the immunosuppressant drug FK506 ameliorate the damage incurred by the 2-h period of focal ischemia, even when given 1-3 h after the start of the recirculation. The primary objective of this study was to find out if FK506, like PBN, prevents the secondary deterioration of mitochondrial function, as this can be studied in vitro. Since this proved to be the case, we addressed the question of whether the secondary mitochondrial dysfunction and bioenergetic failure were related to a secondary compromise of microcirculation and cellular oxygen delivery. Six groups of male Wistar rats were studied for measurement of mitochondrial respiratory activity (total, n = 36). One group was used as control (n = 6). In the other groups of animals, MCA occlusion of 2 h duration was induced by an intraluminal filament technique, Neocortical focal and perifocal ("penumbra") tissues were sampled after 2 h of ischemia (n = 6) and after 1 h (n = 6), 2 h (n = 6 with vehicle), and 4 h (n = 6 with vehicle; n = 6 with FK506) of recirculation. The vehicle or 1.0 mg.kg-1 of FK506 was injected intravenously after 1 h of recirculation. Homogenates were prepared, and stimulated (+ADP), nonstimulated (-ADP), and uncoupled respiratory rates were measured polarographically. The uncoupling agent used was carbonyl cyanide m-chlorophenylhydrazone. Local CBF and tissue oxygen tension were evaluated by laser-Doppler flowmetry and PO2 microelectrodes, respectively, throughout the whole periods of 2 h of ischemia and 4 h of recirculation, using a remote MCA occlusion technique. After 2 h of ischemia, the penumbra showed a moderate decrease and the focus a marked decrease in ADP-stimulated and uncoupled respiratory rates, with a marked fall in the respiratory control ratio, defined as ADP-stimulated divided by nonstimulated respiration. Recirculation (1 h) brought about partial recovery, but continued reflow (2 and 4 h) was associated with a secondary deterioration of respiratory functions. The secondary deterioration was prevented by FK506. The results thus confirm previous findings showing that secondary mi

Topics: Adenosine Diphosphate; Animals; Brain; Cerebrovascular Circulation; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Mitochondria; Oxygen; Oxygen Consumption; Partial Pressure; Rats; Rats, Wistar; 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

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

Many practical therapies have been explored as clinical applications for ischemic cerebral infarction; however, most are still insufficient to treat acute stroke. We show here a potential combination therapy in a rat focal ischemic model to improve neurological symptoms as well as to reduce infarct volumes at the maximum level. We applied protein transduction technology using artificial anti-death Bcl-xl derivative with three amino acid-substitutions (Y22F, Q26N and R165K) (FNK) protein fused with a protein-transduction-domain peptide (PTD-FNK). When PTD-FNK was administrated 1 h after initiating ischemia followed by the administration of an immunosuppressant FK506 with a 30-min time lag, infarct volumes of the total brain and cortex were markedly reduced to 27% and 14%, respectively. This procedure not only reduced the infarct volume and edema, but also markedly improved neurological symptoms. The therapeutic effect continued for at least 1 week after ischemia. FK506 inhibited the transduction of PTD-FNK in vitro, which explains the requirement of a time lag for the administration of FK506. An additional in vitro experiment showed that PTD-FNK, when administered 30 min before FK506, gave the maximal protective effect by reducing the intracellular calcium concentration. We propose that this combination therapy would provide a synergistic protective effect by both drugs, reducing adverse the effects of FK506.

Topics: Animals; Apoptosis; Brain Infarction; Calcium Signaling; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Hypoxia-Ischemia, Brain; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Peptide Fragments; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Tacrolimus; Transduction, Genetic; Treatment Outcome

Transient, global cerebral ischemia (TGCI) leads to hippocampal damage and disruption of spatial learning and memory. The immunosuppressant, tacrolimus (FK506), prevents TGCI-induced hippocampal neurodegeneration, but its effectiveness in promoting the recovery of learning and memory performance after TGCI has been little investigated. Here, we use a confined version of the aversive, non-food rewarded radial maze to evaluate further the effects of FK506 on TGCI-induced learning and memory deficits. In the first experiment, rats were rendered ischemic (15 min 4-VO) and 20 days later were tested for acquisition of the radial maze task over 15 consecutive days (post-operative training). In the second experiment, naive rats were trained for 10 days and subjected to TGCI (pre-operative training); retention of task performance was assessed on days 31, 35 and 39 post-ischemia. Acquisition and retention performances were expressed as a) latency to find a goal box, b) number of reference memory errors, and c) number of working memory errors. Data are presented both across daily training sessions (15 days, 3-day blocks) and as a total value (summed over the 15 days). Histological examination was performed on the day after behavioral testing. In both experiments, FK506 (1.0 mg/kg) was given i.v. at the beginning of reperfusion, followed by doses applied intraperitoneally (i.p.) 6, 24, 48 and 72 h post-ischemia. TGCI markedly disrupted both acquisition and retention performance (p<0.0001-0.05). Treatment with FK506 did not prevent the TGCI-induced acquisition and retention deficits, independently of whether performances were quantified 'daily' or as a 'total' value. In contrast, FK506 reduced hippocampal damage significantly compared to the vehicle alone (p<0.001-0.05). We conclude that the present study did not confirm our earlier behavioral data, and suggest that FK506 is not effective in treating the behavioral outcomes of TGCI, despite its efficacy in reducing CA1, hippocampal damage. However, further studies including other behavioral tasks and more extensive neurohistological analysis, are needed to better elucidate the effectiveness of FK506 in promoting functional recovery in models of transient, global cerebral ischemia.

Topics: Animals; Hippocampus; Immunosuppressive Agents; Ischemic Attack, Transient; Learning Disabilities; Male; Maze Learning; Memory Disorders; Psychomotor Performance; Rats; Rats, Wistar; Tacrolimus

Tacrolimus (FK506) has a neuroprotective action on cerebral infarction produced by cerebral ischemia, however, detailed mechanisms underlying this action have not been fully elucidated. We examined temporal profiles of survival-and death-related signals, Bad phosphorylation, release of cytochrome c (cyt.c), activation of caspase 3 and DNA fragmentation in the brain during and after middle cerebral artery occlusion (MCAo) in mice, and then examined the effect of tacrolimus on these signals. C57BL/6J mice were subjected to transient MCAo by intraluminal suture insertion for 60 min. Tacrolimus (1 mg/kg, i.p.) was administered immediately after MCAo. There were biphasic increases in the release of cyt.c in the ischemic core and penumbra; with the first increase toward the end of the occlusion period and the second increase 3-12 h after reperfusion. Tacrolimus significantly inhibited the increase of cytosolic cyt.c during ischemia and reperfusion. Phosphorylated Bad, Ser-136 (P-Bad(136)) and Ser-155 (P-Bad(155)) were detected 30 min after MCAo and after reperfusion in the ischemic cortex, respectively. Tacrolimus increased P-Bad(136) during ischemia and prolonged P-Bad(155) expression after reperfusion. Tacrolimus also decreased caspase-3 and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling-positive cells, and reduced the size of infarct 24 h after reperfusion. Our study provided the first evidence that the neuroprotective action of tacrolimus involved inhibition of biphasic cyt.c release from mitochondria, possibly via up-regulation of Bad phosphorylation at different sites after focal cerebral ischemia and reperfusion.

Topics: Analysis of Variance; Animals; bcl-Associated Death Protein; Blotting, Western; Brain Infarction; Cytochromes c; Disease Models, Animal; Immunohistochemistry; Immunosuppressive Agents; In Situ Nick-End Labeling; Ischemic Attack, Transient; Male; Mice; Mice, Inbred C57BL; Phosphorylation; Tacrolimus; Time Factors

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

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

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

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

The immunosuppressant FK506 (tacrolimus) exerts potent neuroprotection following focal ischemia in animals; however, the separate effects of FK506 on the ischemic core and penumbra have not been reported. The ischemic penumbra is clinically defined as the difference between a large abnormal area on perfusion-weighted imaging (PWI) and a smaller lesion on diffusion-weighted imaging (DWI). The goal of this study was to determine the effect of FK506 on DWI/PWI match and mismatch areas in transient focal ischemia in rats. Twelve rats were subjected to 1 hr of transient middle cerebral artery (MCA) occlusion, and given an intravenous injection of a placebo (N = 6) or 1 mg/kg FK506 (N = 6) immediately before reperfusion. Magnetic resonance imaging (MRI) was performed during MCA occlusion, and 0.5, 1, and 24 hr after reperfusion. FK506 significantly protected the ischemic brain only in the mismatch cortex where the initial apparent diffusion coefficient (ADC) was normal and there was a mild reduction of cerebral blood flow (CBF). This is the first report to describe the protective effects of FK506 on ischemic penumbra, as measured by DWI/PWI mismatch. The findings provide direct evidence for the utility of DWI/PWI mismatch as a guideline for therapeutic intervention with FK506.

Topics: Animals; Cerebral Cortex; Cerebrovascular Circulation; Corpus Striatum; Diffusion Magnetic Resonance Imaging; Immunosuppressive Agents; Ischemic Attack, Transient; Magnetic Resonance Angiography; Male; Neuroprotective Agents; Rats; Rats, Wistar; Tacrolimus

While the immunosuppressant tacrolimus (FK506) is known to be neuroprotective following cerebral ischemia, the mechanisms underlying its neuroprotective properties are not fully understood. To determine the mode of action by which tacrolimus ameliorates neurodegeneration after transient focal ischemia, we therefore evaluated the effect of tacrolimus on DNA damage, release of cytochrome c, activation of microglia and infiltration of neutrophils following a 60-min occlusion of the middle cerebral artery (MCA) in rats. In this model, cortical brain damage gradually expanded until 24 h after reperfusion, whereas brain damage in the caudate putamen was fully developed within 5 h. Tacrolimus (1 mg/kg) administered immediately after MCA occlusion significantly reduced ischemic damage in the cerebral cortex, but not in the caudate putamen. Tacrolimus decreased both apoptotic and necrotic cell death at 24 h and reduced the number of cytochrome c immunoreactive cells at 8 h after reperfusion in the ischemic penumbra in the cerebral cortex. In contrast, tacrolimus did not show significant neuroprotection for necrotic cell death and reduction of cytochrome c immunoreactive cells in the caudate putamen. Tacrolimus also significantly decreased microglial activation at 8 h and inflammatory markers (cytokine-induced neutrophil chemoattractant and myeloperoxidase [MPO] activity) at 24 h after reperfusion in the ischemic cortex but not in the caudate putamen. These results collectively suggest that tacrolimus ameliorates the gradually expanded brain damage by inhibiting both apoptotic and necrotic cell death, as well as suppressing inflammatory reactions.

Topics: Animals; Apoptosis; Caudate Nucleus; Chemokines, CXC; Cytochromes c; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Ischemic Attack, Transient; Male; Microglia; Neuroprotective Agents; Peroxidase; Rats; Rats, Wistar; Tacrolimus

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

To explore biochemical basis for cerebroprotective effect of immunosuppressant FK506, we studied changes in subcellular distribution of protein kinase C gamma (PKC gamma) as well as calcium/calmodulin-dependent protein kinase II (CaMKII) after ischemia. Male Mongolian gerbils were subjected to 5 min forebrain ischemia. FK506 (1 or 3 mg kg-1) was administered at 1 min after recirculation, which was confirmed to be cerebroprotective by histological examination at seven days after ischemia. At the designated time points (before ischemia, 5 min ischemia, 1 and 24 h recovery), heads were frozen and samples were taken from CA1 subfield of hippocampus. Western blot analysis was carried out. Persistent translocations of PKC gamma and CaMKII to synaptosomal P2 fraction were observed in vehicle-treated group. FK506 significantly decreased levels of PKC gamma and CaMKII in P2 fraction at 24 h of recovery. The present results suggest FK506 downregulates translocated PKC gamma and CaMKII, which may contribute to its survival promoting effect after cerebral ischemia.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Gerbillinae; Hippocampus; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Neurons; Neuroprotective Agents; Protein Kinase C; Tacrolimus

The authors evaluated the therapeutic efficacy of tacrolimus (FK506), administered alone or in combination with recombinant tissue plasminogen activator (t-PA), on brain infarction following thrombotic middle cerebral artery (MCA) occlusion. Thrombotic occlusion of the MCA was induced by a photochemical reaction between rose bengal and green light in Sprague-Dawley rats, and the volume of ischemic brain damage was determined 24 hours later. Intravenous administration of tacrolimus or t-PA dose-dependently reduced the volume of ischemic brain infarction, whether administered immediately or 1 hour after MCA occlusion. When tacrolimus or t-PA was administered 2 hours after MCA occlusion, each drug showed a tendency to reduce ischemic brain damage. However, combined treatment with both drugs resulted in a significant reduction in ischemic brain damage. On administration 3 hours after MCA occlusion, tacrolimus alone showed no effect, and t-PA tended to worsen ischemic brain damage. However, the combined treatment with both drugs not only ameliorated the worsening trend seen with t-PA alone, but also tended to reduce ischemic brain damage. In conclusion, tacrolimus, used in combination with t-PA, augmented therapeutic efficacy on brain damage associated with focal ischemia and extended the therapeutic time window compared to single-drug treatments.

Topics: Animals; Blood Flow Velocity; Cerebrovascular Circulation; Disease Models, Animal; Drug Therapy, Combination; Intracranial Thrombosis; Ischemic Attack, Transient; Male; Middle Cerebral Artery; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tacrolimus; Time Factors; Tissue Plasminogen Activator

The two immunosuppressants, cyclosporin A (CsA) and FK506, when given 1 and 3 h after the start of reperfusion following 2 h of middle cerebral artery (MCA) occlusion, reduce infarct volume to 30% of control. This suggests a common effect, e.g. one due to suppression of the activation of calcineurin. However, when given by the intracarotid (i.c.) route after only 5 min of recirculation CsA, but not FK506, reduced infarct volume even further, to 10% of control. This was attributed to the fact that CsA, but not FK506, block the in vitro assembly of a mitochondrial permeability transition (MPT) pore. The present experiments were undertaken to further characterize the anti-ischemic effect of CsA, when given i.c. 5 min after recirculation and to explore why CsA, when given at that time, is more efficacious than FK506. It was established that the i.c. administration of CsA in a dose of 10 mg/kg increased the tissue concentration of CsA 2- to 3-fold, when compared to the i.v. administration. CsA proved to be effective in reducing infarct volume even when the tissue damage was assessed by histopathology after 7 days of recovery. MCA occlusion of 2 h duration caused a sustained decrease in the phosphorylation Akt at threonine 308. Since this down regulation of Akt was prevented by CsA, the results suggested a link between dephosphorylaltion of Bad, and cell death. Interestingly FK506 did not prevent down regulation of Akt, it thus seems unlikely that the anti-ischemic effect of CsA is related to its association with cytosolic cyclophilin.

Topics: Animals; Brain; Cyclosporine; Down-Regulation; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; 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

The protective effect of the immunosuppressant agent FK506 in the reperfusion after short-term occlusion of the middle cerebral artery in the rat model was evaluated using [125I]PK-11195 autoradiography. FK506 0.5 mg kg-1 day-1 was administered intramurally to Wistar rats weighing 260-300 g from one day prior to ischemia to seven days after ischemia. Reperfusion was performed after 30 or 60 min occlusion. Infarct area was evaluated by [125I]PK-11195 autoradiography on the seventh day following occlusion. FK506 significantly reduced the infarct area in the caudate nucleus following 30 and 60 min occlusion, but significantly reduced the infarct area in the cortex only following 60 min occlusion. These results suggest that FK506 has a protective effect against reperfusion after short-term occlusion of the middle cerebral artery.

Topics: Animals; Autoradiography; Cerebral Infarction; Disease Models, Animal; Iodine Radioisotopes; Ischemic Attack, Transient; Isoquinolines; Male; Middle Cerebral Artery; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion; Tacrolimus

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

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

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

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

We investigated the changes in the enzyme activity and immunoreactivity of calcineurin in the rat hippocampus after transient forebrain ischemia. Immediately after 20-min transient forebrain ischemia, calcineurin activity decreased to about 40% of the control in the CA1 region and to about 55% in other regions. Protein phosphatase 2A activity showed no remarkable changes. By 12 h after ischemia, calcineurin activity recovered, more in the CA1 region than in other regions. At 24 h it decreased again, but only in the CA1 region. Immunohistochemical- and immunoblot analyses showed no remarkable change in calcineurin in any region of the hippocampus within 12 h after ischemia. Thus, the activity of calcineurin is dissociated from its immunoreactivity and quantity. Several studies have suggested that unknown inhibitory factor(s) and/or reversible changes in calcineurin act to modify enzyme activity after ischemia. In contrast, phosphatase 2A activity underwent no obvious changes during the post-ischemia period we examined. This unique time course of calcineurin activity may contribute to the mechanism of ischemic neuronal injury.

Topics: Animals; Calcineurin; Dentate Gyrus; Enzyme Activation; Ferrous Compounds; Immunoblotting; Immunohistochemistry; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Phosphoprotein Phosphatases; Prosencephalon; Protein Phosphatase 2; Rats; Rats, Wistar; Subcellular Fractions; Tacrolimus

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

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

In the present experiments, we compared the anti-ischemic effects of the immunosuppressants cyclosporin A (CsA) and FK506 in hyperglycemic animals subjected to 30 min of middle cerebral artery (MCA) occlusion. Both immunosuppressants were given as pre-treatment, the effect of treatment being evaluated by 2,3, 5-triphenyltetrazolium (TTC) staining after 3 days of recovery. Both FK506 and CsA reduced the infarct volume to less than 1/3 of control. In spite of CsA's known effect as a blocker of the mitochondrial transition (MPT) pore, it failed to give a more robust effect than FK506. If anything, FK506, which lacks an effect on the MPT pore, had a more pronounced anti-ischemic effect. We conclude that, in this model of infarction, an MPT may not play a major pathogenetic role.

Topics: Analysis of Variance; Animals; Arterial Occlusive Diseases; Cerebral Infarction; Cyclosporine; Hyperglycemia; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Neuroprotective Agents; Rats; Rats, Wistar; Tacrolimus; Treatment Outcome

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

The effect of FK 506, an immunosuppressant which is widely used in the transplantation of liver, kidney and bone marrow, on cerebral infarction volume in transient focal ischemia was investigated. Focal cerebral ischemia was produced by 2 h occlusion of the left middle cerebral artery in rats. FK 506 (0.3 mg/kg body weight) and vehicle were administered intravenously immediately after induction of cerebral ischemia. After 24 h reperfusion the rats were sacrificed and brain infarct volume and edema volume were determined. The FK 506 treatment group showed a significantly reduced infarct volume in the cerebral cortex when it was compared with the vehicle treatment group, but infarct volume was not significantly reduced in the striatum which was the ischemic core in this focal ischemia model. However, FK 506 did not reduce the edema volume significantly. These results suggest that the reduction of infarct volume is a result of the neuroprotective effect of FK 506. This immunosuppressant may be useful in the treatment of cerebral infarction.

Topics: Animals; Cerebral Cortex; Corpus Striatum; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Rats; Rats, Sprague-Dawley; Tacrolimus

Delayed neuronal death (DND) in CA1 region after transient global ischemia is a well-known phenomenon, but its mechanism has not been clarified. In order to examine the involvement of nitric oxide (NO) in DND, 7-nitro indazole (7NI), a selective neuronal NO synthase (nNOS) inhibitor, and FK506, an immunosuppressant which also inhibits nNOS, were administered intraperitoneally during and after transient global ischemia in gerbil. FK506 moderately ameliorated DND in a dose-dependent manner. However, 7NI showed only minor neuroprotective effects. These results show that DND is not mainly mediated by NO production via nNOS, and FK506 acts as a neuroprotective agent via unknown pathways other than nNOS inhibition.

Topics: Animals; Cell Death; Enzyme Inhibitors; Gerbillinae; Immunosuppressive Agents; Indazoles; Ischemic Attack, Transient; Male; Neurons; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase; Prosencephalon; Tacrolimus

To evaluate the effect of FK506 on delayed neuronal death in gerbils after forebrain ischemia, 84 adult Mongolian gerbils were used in this study. Transient forebrain ischemia was induced by clipping common carotid arteries bilaterally for 5 minutes. One hour after reperfusion we intraperitoneally injected FK506 (1.0 mg/kg), cyclosporin A (CsA) (10.0 mg/kg) or the vehicle solution into each gerbil. In one group, each agent was additionally administered daily 3 more times at 24, 48 and 72 hours after ischemia. The gerbils were killed 4 days or 10 days after transient ischemia, and damage to their hippocampal pyramidal cells was histologically assessed. Additionally, the body temperature was measured following administration of each drug to investigate drug-induced hypothermia. Post-ischemic repeated treatment with FK506 significantly (p < 0.01) reduced degeneration of hippocampal neurons. However, partial treatment did not modify neuronal degeneration. CsA did not show a neuroprotective effect in this study. Drug-induced mild hypothermia (35-37 C) was observed following administration of FK506 or CsA. There was no significant difference in the time course of the body temperature between the FK506 and CsA group. We demonstrated that the repeated FK506 treatment, but not the CsA treatment, reduced ischemia-induced degeneration of hippocampal neurons in gerbils. Although FK506-induced hypothermia might have modified neuronal degeneration, a comparison with CsA indicated that the neuroprotective effect of FK506 was not solely due to hypothermia per se.

Topics: Animals; Body Temperature; Cell Death; Cyclosporine; Female; Gerbillinae; Hippocampus; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Nerve Degeneration; Neuroprotective Agents; Pyramidal Cells; Tacrolimus

We hypothesized that the immunological reaction against extravasated blood might play a role in the development of vasospasm after subarachnoid hemorrhage. Under the hypothesis, we had reported significant therapeutic efficacy of cyclosporin A on vasospasm in canine models. We here investigated the efficacy of a new, potent immunosuppressant, FK-506, on vasospasm in animal models. Dogs were randomly classified into sham operated, subarachnoid hemorrhage treated-1 group, (FK-506, 0.3 mg/kg-d, intramuscular injection), and treated-2 group (FK-506, 0.15 mg/kg-d, intramuscular injection). Levels of the third factor of complement (C3) and the activity of serum complement inducing 50% hemolysis of sheep erythrocytes (CH50) in serum were also determined. In the treated groups, the levels of FK-506 in serum were monitored. As for C3 and CH50, there were no statistically significant differences among the groups and there were no changes between Day 1 and Day 7 in any group. Angiographical diameters of a basilar artery on Days 1 and 7 were measured with a computed image analyzer, and the extent of vasospasm was compared among the groups. Statistically significant differences between the sham-operated group and the other three groups were noted. However, under sufficient levels of FK-506 in serum, the extent of vasospasm in either treated group was the same as that in the subarachnoid hemorrhage group. These results indicate a significant discrepancy in the therapeutic mechanism for vasospasm between cyclosporin A and FK-506. They have common aspects in the immunosuppressive mechanism. However, in T-cell suppression, the different mechanism in situ between the two drugs is also postulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cerebral Angiography; Complement C3; Dogs; Female; Ischemic Attack, Transient; Male; Muscle, Smooth, Vascular; Subarachnoid Hemorrhage; Tacrolimus

To define the relationship between the immunologic reaction and the pathogenesis of cerebral vasospasm (VS) following experimental subarachnoid hemorrhage (SAH), we examined the effect of a cell mediated immunosuppressive agent, FK-506, isolated from Streptomyces tsukubaensis, by using the canine SAH model. There was a significant vasoconstriction in the basilar artery in the control group after SAH. This constriction, however was not successfully prevented by FK-506 or combination of FK-506 and steroid, since there was no significant difference in the vessel caliber size among these groups. The pathologic approach, accompanied by immunohistochemistry, could not discriminate the differences in the nature of the lesion between the untreated group and FK-506 treated groups, except for slight lymphocytic infiltrations present around the basilar artery of untreated group. Histopathologically, inflammatory reactions consisting of neutrophils, that were not suppressed by FK-506 treatment, were clearly seen around the spastic vessels in the subarachnoid space. Furthermore, several constrictive changes or degenerative alterations were also observed in the spastic vascular wall. Immunohistochemically, the deposition of IgG, IgM and C3 was present in the intima and the luminal side of the smooth muscle layer, and capillary vessels of the brain stem. It is considered that this deposition was caused by increased vascular permeability in VS. On the basis of the above findings that the cell mediated immunosuppressive agent, FK-506 failed to prevent vasoconstriction or pathologic lesions but lymphocytic infiltrations, it is considered that the cell mediated immunopathogenesis may play little role in producing VS following SAH.

Topics: Animals; Basilar Artery; Brain Stem; Dogs; Female; Ischemic Attack, Transient; Male; Muscle, Smooth, Vascular; Subarachnoid Hemorrhage; Tacrolimus

In order to clarify the possible role of immunological reaction in the pathogenesis of cerebral vasospasm, the authors examined the prophylactic effect of the immunosuppressant agents FK-506 and cyclosporin A on chronic vasospasm in a canine two-hemorrhage model. While a mean constriction of the basilar artery to 81.0% +/- 4.0% (+/- standard error of the mean) occurred on Day 2 and to 63.8% +/- 3.5% on Day 7 in the untreated group, constriction to 77.9% +/- 3.4% on Day 2 and 62.8% +/- 3.0% on Day 7 was demonstrated in the FK-506-treated group (difference not significant). This tendency was also noted in the cyclosporin A-treated group, with basilar artery constriction to 81.8% +/- 3.7% and 56.3% +/- 2.7%, respectively (difference not significant). The histological changes of the basilar artery, including corrugation of the elastic lamina, detachment of endothelial cells, and vacuolar formation in the smooth-muscle layer were not different in the two treated groups and the one control group. Since these immunosuppressant agents are known to inhibit the release of interleukin-2 (IL-2), the level of IL-2 was examined in the cerebrospinal fluid of patients with cerebral vasospasm. While interleukin-1 gradually increased in level as time passed, the level of IL-2 was consistently low during the course of the study, indicating less participation of IL-2 in the pathogenesis of cerebral vasospasm. This clinical observation matched the experimental results. The authors conclude that cell-mediated immunoreaction, initiated mainly by IL-2, plays little role in the pathogenesis of cerebral vasospasm.

Topics: Animals; Cyclosporine; Dogs; Immunosuppressive Agents; Interleukin-2; Ischemic Attack, Transient; Subarachnoid Hemorrhage; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Tacrolimus