Yasuhisa Furuichi

Neuroprotective action of tacrolimus (FK506) in focal and global cerebral ischemia in rodents: dose dependency, therapeutic time window and long-term efficacy.

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.

Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia

Adult neurogenesis is modulated by growth factors, physical conditions, and other alterations in the physical microenvironment. We studied the effects of focal ischemia on neurogenesis in the subventricular zone (SVZ), olfactory bulb (OB), and hippocampal dentate gyrus (DG) (known to be persistent neurogenic regions) in the adult non-human primate, the cynomolgus monkey. Three monkeys underwent middle cerebral artery occlusion-induced focal ischemia and were given multiple BrdU injections during the first 2 weeks after ischemia. Twenty-eight days later, the animals were perfused. The number of new neurons (3182 +/- 408/mm3) in the ipsilateral DG of ischemic monkeys was 4.7-fold that in the DG of non-operated monkeys. The number of new neurons (9176 +/- 2295/mm3) in the ipsilateral olfactory bulb of ischemic monkeys was 18.0-fold that in normal olfactory bulb. These observations suggest an increase in the number of new OB neurons, as well as new DG neurons, after focal ischemia in a primate. This substantial increase in new neurons after focal ischemia could result from the enhancement of cell proliferation rather than a change in the rate of cell commitment. Of the three monkeys subjected to ischemia, only one animal possessed a unique progenitor cell type at the most anterior aspect of the ipsilateral SVZ. Within this region, a short migration (approximately 500 microm) of doublecortin-expressing immature neuronal progenitor cells was observed.

A Combined Treatment with Tacrolimus (FK506) and Recombinant Tissue Plasminogen Activator for Thrombotic Focal Cerebral Ischemia in Rats: Increased Neuroprotective Efficacy and Extended Therapeutic Time Window

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.

Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats.

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.

Tacrolimus, a Potential Neuroprotective Agent, Ameliorates Ischemic Brain Damage and Neurologic Deficits after Focal Cerebral Ischemia in Nonhuman Primates

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.

Therapeutic time window of tacrolimus (FK506) in a nonhuman primate stroke model: comparison with tissue plasminogen activator.

Tacrolimus (FK506), an immunosuppressive drug, has been shown to exert a potent neuroprotective activity when administered immediately after occlusion of the middle cerebral artery (MCA) in a nonhuman primate model of stroke. Here, we assessed the neuroprotective efficacy of tacrolimus with delayed treatment using the same model and compared with that of recombinant tissue plasminogen activator (rt-PA). Ischemic insult was induced by photochemically induced thrombotic occlusion of MCA in cynomolgus monkeys, and tacrolimus (0.2 mg/kg) and/or rt-PA (1.0 mg/kg) was intravenously administered 2 h after MCA occlusion. In another experiment, tacrolimus (0.1 mg/kg) was administered 4 h after MCA occlusion. Neurological deficits were monitored for 28 days after the ischemic insult and cerebral infarct volumes were measured with brain slices. With drug administration 2 h after the ischemic insult, tacrolimus significantly reduced neurological deficits and infarct volumes in the cerebral cortex without affecting the recanalization pattern in the MCA, however, rt-PA did not significantly improve neurological deficits or infarct volumes, even though it increased the recanalization rate of the occluded MCA. Combined treatment with tacrolimus and rt-PA exerted additional protection. Administration of tacrolimus 4 h after the ischemic insult still showed significant amelioration of neurological deficits. These results suggested that tacrolimus had a wider therapeutic time window than rt-PA in the nonhuman primate stroke model.

Neuroprotective efficacy of FR901459, a novel derivative of cyclosporin A, in in vitro mitochondrial damage and in vivo transient cerebral ischemia models.

The immunosuppressant cyclosporin A (CsA) has been shown to exert potent neuroprotective effects, possibly via the inhibition of calcineurin and mitochondrial permeability transition pore formation. Here, we investigated the neuroprotective profile of a novel derivative of CsA, FR901459, by evaluating its effects against in vitro mitochondrial damage and in vivo brain damage in transient global or focal cerebral ischemia models, in comparison with those of CsA. Efficacy of calcineurin inhibition was estimated from its immunosuppressive effect on the mixed lymphocyte reaction. Results showed that the immunosuppressive effect of FR901459 was approximately 7-fold less potent than that of CsA. In contrast, FR901459 suppressed Ca(2+)-induced mitochondrial swelling measured in isolated liver mitochondria with greater potency than CsA. Further, FR901459 showed approximately 30-fold greater neuroprotective potency than CsA against neuronal cell damage induced by thapsigargin in SH-SY5Y cells. In a transient global cerebral ischemia model in gerbils, FR901459 showed the dose-dependent suppression of neuronal cell death, while FR901459 was less efficacious than CsA. In a rat transient focal ischemia model, FR901459 tended to reduce brain damage on both intravenous injection as well as intracerebroventricular infusion, but with less efficacy than CsA which significantly reduced the damage. These findings suggest that FR901459 exerts a potent neuroprotective effect by inhibiting mitochondrial damage in vitro, but that in in vivo transient cerebral ischemia, its immunosuppressive component which possibly acts via the inhibition of calcineurin may play a more important role in attenuating brain damage than its inhibitory effect against mitochondrial damage.

Characterization of a novel thrombotic middle cerebral artery occlusion model in monkeys that exhibits progressive hypoperfusion and robust cortical infarction.

In an attempt to establish a thrombotic middle cerebral artery (MCA) occlusion model using cynomolgus monkeys, we measured the blood flow in the main MCA tract and cerebral cortex, brain damage, and neurological deficits, and compared them with those of mechanical MCA occlusion model. Thrombotic occlusion was induced photochemically by green light application on the MCA following rose bengal treatment; mechanical occlusion was induced by MCA clipping for 3h. Patency of the main MCA tract showed two patterns in the thrombotic model: permanent occlusion or cyclical flow reduction (CFR). Regional cerebral blood flow (rCBF) decreased during occlusion followed by post-ischemic hyperperfusion in the clipping model, whereas rCBF reduction expanded time-dependently in the thrombotic occlusion model. Brain infarction and neurological scores in the thrombotic occlusion model were significantly larger than those in the clipping occlusion model. In histological assessment, microthrombi containing myeloperoxidase- and fibrinogen-positive cells were observed in the cortex following the thrombotic but not clipping occlusion. These results collectively suggest that this thrombotic MCA occlusion model, because it shows impairment of cerebral microcirculation, could provide a vital platform for understanding progressive ischemia as well as for evaluating potential therapeutic drugs.

Tacrolimus (FK506) attenuates biphasic cytochrome c release and Bad phosphorylation following transient cerebral ischemia in mice

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.

Tacrolimus (FK506) suppresses rt-PA-induced hemorrhagic transformation in a rat thrombotic ischemia stroke model

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.