Chikako Nito

Neuroprotective effect of immunosuppressant FK506 in transient focal ischemia in rats: Therapeutic time window for FK506 in transient focal ischemia

Abstract 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 0.3mg 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. [Neurol Res 2001; 23: 755-760]

Mild hypothermia enhances the neuroprotective effects of FK506 and expands its therapeutic window following transient focal ischemia in rats

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.

The effect of ascorbic acid on the pharmacokinetics of levodopa in elderly patients with Parkinson disease

Levodopa (LD) is one of the most effective drugs for clinical symptoms in patients with Parkinson disease (PD). Most PD patients are advanced in age and may have trouble with LD absorption because aging influences drug absorption processes. Previous reports have indicated that ascorbic acid (AsA) can reduce LD dosage without losing its effectiveness. The current study sought to determine whether AsA affects LD absorption in elderly PD patients. Sixty-seven elderly PD patients took a tablet orally containing 100 mg LD and 10 mg carbidopa following an overnight fast. Plasma LD concentrations were determined at 6 points up to 3 hours, using high-performance liquid chromatography with electrochemical detection. The area under the curve (AUC), peak drug concentration (Cmax), and time to peak drug concentration (Tmax) were calculated. The pharmacokinetic evaluation was repeatedly performed 1 week later in the same way except for adding 200 mg AsA to the tablet. The changes in AUC, Cmax, and Tmax between the tests were evaluated. Significant changes in these parameters were not observed when analyzed using data from all patients. However, significant increases in AUC and Cmax, and a significant reduction in Tmax by adding AsA were observed in 25 patients with baseline AUC ≤2500 ng·hour/mL. In conclusion, AsA can improve LD absorption in elderly PD patients with poor LD bioavailability. LD therapy in combination with AsA may be one of the strategies for PD treatment.

FK506 ameliorates oxidative damage and protects rat brain following transient focal cerebral ischemia

Abstract Objective: 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. Methods: 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. Results: 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). Discussion: These results demonstrate that FK506 may have antioxidant as well as anti-inflammatory effects and reduces ischemic damage following cerebral infarction.

Therapeutic impact of eicosapentaenoic acid on ischemic brain damage following transient focal cerebral ischemia in rats.

Long-chain n-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have been shown to reduce ischemic neuronal injury. We investigated the effects of ethyl-EPA (EPA-E) on ischemic brain damage using a rat transient focal cerebral ischemia model. Male Sprague-Dawley rats (n=105) were subjected to 90 min of focal cerebral ischemia. EPA-E (100mg/kg/day) or vehicle was administered once a day for 3, 5 or 7 days prior to ischemia. Different withdrawal intervals of 3, 5, and 7 days prior to ischemia following 7-day pretreatment with EPA-E or vehicle were also examined. In addition, post-ischemic administration of EPA-E was investigated. Pretreatment with EPA-E for 7 and 5 days, but not 3 days, showed significant infarct volume reduction and neurological improvements when compared with vehicle pretreatment. In addition, withdrawal of EPA-E administration for 3 days, but not 5 and 7 days, also demonstrated significant infarct volume reduction and neurological improvements when compared with vehicle treatment. Post-ischemic treatment of EPA-E did not show any neuroprotection. Immunohistochemistry revealed that 7-day pretreatment with EPA-E significantly reduced cortical expression of 8-hydroxydeoxyguanosine (maker for oxidative DNA damage), 4-hydroxy-2-nonenal (maker for lipid peroxidation), phosphorylated adducin (marker for Rho-kinase activation) and von Willebrand factor (endothelial marker) when compared with vehicle pretreatment. In addition, phosphorylated adducin expression co-localized with von Willebrand factor immunoreactivity. The present study established the neuroprotective effect of EPA-E on ischemic brain damage following transient focal cerebral ischemia in rats, which may be involved in the suppression of oxidative stress and endothelial Rho-kinase activation.

Continuous oral administration of atorvastatin ameliorates brain damage after transient focal ischemia in rats

AIMS Pre-treatment with statins is known to ameliorate ischemic brain damage after experimental stroke, and is independent of cholesterol levels. We undertook pre- vs post-ischemic treatment with atorvastatin after focal cerebral ischemia in rats. MAIN METHODS Male Sprague-Dawley rats underwent transient 90-min middle cerebral artery occlusion (MCAO). Atorvastatin (20mg/kg/day) or vehicle was administered orally. Rats were divided into vehicle-treated, atorvastatin pre-treatment, atorvastatin post-treatment, and atorvastatin continuous-treatment groups. In the pre-treatment, rats were given atorvastatin or vehicle for 7 days before MCAO. In the post-treatment, rats received atorvastatin or vehicle for 7 days after MCAO. Measurement of infarct volume, as well as neurological and immunohistochemical assessments, were done 24h and 7 days after reperfusion. KEY FINDINGS Each atorvastatin-treated group demonstrated significant reductions in infarct and edema volumes compared with the vehicle-treated group 24h after reperfusion. Seven days after reperfusion, infarct volumes in the post-treatment group and continuous-treatment group (but not the pre-treatment group) were significantly smaller than in the vehicle-treated group. Only the continuous-treatment group had significantly improved neurological scores 7 days after reperfusion compared with the vehicle group. Post-treatment and continuous-treatment groups had significantly decreased lipid peroxidation, oxidative DNA damage, microglial activation, expression of tumor necrosis factor-alpha, and neuronal damage in the cortical ischemic boundary area after 7 days of reperfusion. SIGNIFICANCE These results suggest that continuous oral administration (avoiding withdrawal) with statins after stroke may reduce the extent of post-ischemic brain damage and improve neurological outcome by inhibiting oxidative stress and inflammatory responses.

Valproic acid ameliorates ischemic brain injury in hyperglycemic rats with permanent middle cerebral occlusion.

Valproic acid (VPA) is widely used for the clinical treatment of epilepsy. Previous studies have demonstrated that VPA ameliorates brain injury following experimental stroke. However, the effect of VPA in stroke models featuring comorbid conditions has not been fully explored. In this study, we investigate the effects of VPA on permanent ischemic stroke with hyperglycemia. Hyperglycemia was induced by streptozotocin (STZ) injection 3 days before. Test animals received a single injection of VPA immediately after induction of ischemia. Control animals received occlusion and physiological saline injection, or STZ, occlusion, and saline. Magnetic resonance imaging of cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) was performed 60 min after ischemia. Infarct volume, neurological deficits, rotarod test performance, and immunohistological markers were assessed 3 days after ischemia. Hyperglycemia significantly expanded the area of decreased of CBF and ADC, and increased the number of myeloperoxidase-positive cells, ionized calcium binding adapter molecule 1-positive cells, inducible nitric oxide synthase-positive cells, von Willebrand factor-positive cells, and Fluoro-Jade C-positive cells in the ischemic boundary zone, which was accompanied by increased infarct volume and deteriorated neurological deficit and rotarod test compared with normoglycemia (P < 0.05). VPA significantly alleviated the aggravation of functional outcome accompanied by suppressing these inflammation, endothelial injury, and neuronal degeneration compared with saline-treated group (P < 0.05). A single injection of VPA following permanent ischemia in STZ-induced hyperglycemic rats ameliorates neurological deficits and reduces neuronal degeneration by inhibiting inflammation and endovascular injury. VPA may be promising as a candidate therapy for human stroke.

Low free triiodothyronine predicts poor functional outcome after acute ischemic stroke

BACKGROUND AND PURPOSE The aim of this study was to investigate the association of admission serum thyroid hormone concentration with clinical characteristics and functional outcomes in patients after acute ischemic stroke. METHODS We retrospectively enrolled 398 consecutive patients admitted to our stroke center between July 2010 and April 2012. Serum thyroid stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) were evaluated upon admission. Neurological severity was evaluated using the National Institutes of Health Stroke Scale (NIHSS) upon admission and the modified Rankin Scale (mRS) upon discharge. Poor outcome was defined as a mRS score of 3-5 or death (mRS score 6). Separate analyses were conducted according to outcome and quartile serum FT3 concentration. RESULTS In total, 164 patients (41.2%) demonstrated a poor outcome. Age, male gender, blood glucose level, arterial fibrillation, dyslipidemia, smoking, NIHSS score, cardioembolic stroke type, and periventricular hyperintensities, but not FT4 or TSH, were significantly associated with poor functional outcome. Furthermore, poor functional outcome was independently associated with low FT3 (<2.29pg/mL). In comparisons between FT3 quartiles (Q1 [≤2.11pg/mL], Q2 [2.12-2.45pg/mL], Q3 [2.46-2.77pg/mL], Q4 [≥2.78pg/mL]), patients with poor outcomes were more frequent in Q1 than in Q4 after multivariate adjustment. Death was more frequent in Q1 than in Q4 after adjustment for risk factors and comorbidities, but this difference was non-significant after additional adjustment for age and NIHSS score. CONCLUSIONS Our data suggest that a lower FT3 value upon admission may predict a poor functional outcome in patients with acute ischemic stroke. Further large-scale prospective studies are required to clarify the role of thyroid hormone in the acute phase of ischemic stroke.

Initial Symptoms of Parkinson’s Disease with Elderly Onset

Background/Objective: As the incidence of Parkinson’s disease (PD) is related to aging, we consider it important to determine how the initial symptoms change with age in order to diagnose early elderly cases of PD accurately. Methods: 84 patients (age at onset 70.7 ± 9.0 years; mean ± 1 SD) were studied to see whether the initial symptoms change according to age. Results: The prevalence of resting tremor was significantly lower in patients of advanced age (p = 0.041). In contrast, the incidence of postural and gait disorders increased significantly with aging (p = 0.032). The prevalences of rigidity and kinetic disorders, which are important clinical features of PD, were not influenced by aging. Conclusion: These findings suggest that the cause of PD is not related to the aging process itself, since the prevalences of all symptoms were not influenced by aging. Knowledge of the prevalence of the inital symptoms of PD may contribute to the accurate diagnosis in early and elderly cases.

Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke

Interleukin (IL)-10 is a contributing factor to neuroprotection of mesenchymal stem cell (MSC) transplantation after ischemic stroke. Our aim was to increase therapeutic effects by combining MSCs and ex vivo IL-10 gene transfer with an adeno-associated virus (AAV) vector using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague-Dawley rats underwent 90 min MCAO followed by intravenous administration of MSCs alone or IL-10 gene-transferred MSCs (MSC/IL-10) at 0 or 3 hr after ischemia reperfusion. Infarct lesions, neurological deficits, and immunological analyses were performed within 7 days after MCAO. 0-hr transplantation of MSCs alone and MSC/IL-10 significantly reduced infarct volumes and improved motor function. Conversely, 3-hr transplantation of MSC/IL-10, but not MSCs alone, significantly reduced infarct volumes (p < 0.01) and improved motor function (p < 0.01) compared with vehicle groups at 72 hr and 7 days after MCAO. Immunological analysis showed that MSC/IL-10 transplantation significantly inhibits microglial activation and pro-inflammatory cytokine expression compared with MSCs alone. Moreover, overexpressing IL-10 suppressed neuronal degeneration and improved survival of engrafted MSCs in the ischemic hemisphere. These results suggest that overexpressing IL-10 enhances the neuroprotective effects of MSC transplantation by anti-inflammatory modulation and thereby supports neuronal survival during the acute ischemic phase.