Toshiki Inaba

Dissociation and protection of the neurovascular unit after thrombolysis and reperfusion in ischemic rat brain

In the ischemic brain, reperfusion with tissue plasminogen activator (tPA) sometimes causes catastrophic hemorrhagic transformation (HT); however, the mechanism remains elusive. Here, we show that the basement membrane, and not the endothelial cells, is vulnerable to ischemic/reperfusion injury with tPA treatment. We treated a spontaneously hypertensive rat model of middle cerebral artery occlusion (MCAO) with vehicle alone, tPA alone, or a free radical scavenger, edaravone, plus tPA. Light and electron microscopic analyses of each microvascular component revealed that the basement membrane disintegrated and became detached from the astrocyte endfeet in tPA-treated animals that showed HT. On the other hand, edaravone prevented the dissociation of the neurovascular unit, dramatically decreased the HT, and improved the neurologic score and survival rate of the tPA-treated rats. These results suggest that the basement membrane that underlies the endothelial cells is a key structure for maintaining the integrity of the neurovascular unit, and a free-radical scavenger can be a viable agent for inhibiting tPA-induced HT.

Intra-arterial transplantation of bone marrow mononuclear cells immediately after reperfusion decreases brain injury after focal ischemia in rats

AIMS Transplantation of bone marrow cells has been reported to exert neuroprotection against cerebral ischemia. However, the effect of bone marrow mononuclear cells (BMMCs) administered immediately after reperfusion has rarely been investigated. The present study was designed to examine whether brain injury in response to transient focal ischemia can be ameliorated by BMMC administration immediately after reperfusion in rats, and to determine whether there are differences in the route of administration. MAIN METHODS Autologous BMMCs were obtained from each rat. Rats were then subjected to transient focal ischemia followed by BMMC administration via the ipsilateral carotid artery (IA group) or the femoral vein (IV group) immediately after reperfusion. Control rats underwent the same procedure but received vehicle injection. Infarct volume was compared among the groups 24 h and 7 days after reperfusion. BMMCs were fluorescently labeled with PKH26 prior to administration to track transplanted cells. KEY FINDINGS Total infarct volume decreased in the IA group, but not in the IV group, when compared to the vehicle group. In the ipsilateral hemisphere, PKH26 positive cell count was greater in the IA group than in the IV group. Motor function, assessed with a rotarod test, improved in the IA group compared to the vehicle group. SIGNIFICANCE These results show significant neuroprotection after transient focal ischemia by 1 x 10(7) autologous BMMCs administered intra-arterially, but not intravenously, immediately after reperfusion in rats. The larger number of transplanted BMMCs in the brain during the early stage of reperfusion may be responsible for the protective effect.

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.

Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration of ischemic brain damage in rats

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

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.

Neuroprotective effects of erythromycin on cerebral ischemia reperfusion-injury and cell viability after oxygen-glucose deprivation in cultured neuronal cells.

This study aims to determine if erythromycin has neuroprotective effects against transient ischemia and oxygen-glucose deprivation (OGD) in cultured neuronal cells. Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 90 min, followed by reperfusion. The animals received a subcutaneous single injection of erythromycin lactobionate (EM, 50mg/kg) or vehicle immediately after ischemia. Infarct volume, edema index, and neurological performance were evaluated at 24 and 72 h after reperfusion. Immunohistochemical analyses for oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) were conducted in the cortex at 24h. Primary cortical neuronal cell cultures were prepared from the cerebral cortices of the animals and then subjected to OGD for 3h. Ten or 100 μM EM was added before OGD to determine the effect of EM on cell viability after OGD. EM significantly reduced infarct volume (p<0.01) and edema volume (p<0.05) and improved neurological deficit scores (p<0.05) at 24 and 72 h. EM significantly suppressed the accumulation of 4-HNE (p<0.01) and 8-OHdG (p<0.01) and markedly reduced Iba-1 (p<0.01) and TNF-α expression (p<0.01). Treatment with 100 μM EM in vitro significantly reduced cell death after OGD. EM reduces neuronal damage following cerebral ischemia and OGD and may have antioxidant and anti-inflammatory effects.

The effect of cilostazol and aspirin pre-treatment against subsequent transient focal cerebral ischemia in rat

Abstract Objectives: Among several anti-platelet drugs to prevent recurrent stroke, cilostazol has shown various effects besides its anti-platelet activity. We examined whether 7 days of oral administration of cilostazol protects against subsequent cerebral ischemia, and whether or not the effect of combination therapy with aspirin is more protective. Methods: We used Sprague-Dawley (SD) rats and assigned them to four groups: vehicle, aspirin, cilostazol, and aspirin plus cilostazol combination therapy. After oral administration of anti-platelets for 7 days, we performed transient middle cerebral artery occlusion (MCAO) for 90 minutes, and examined infarct volume, neurological symptoms, and regional cerebral blood flow (rCBF). Immunostaining of Bax, Bcl-2, TUNEL, 4-HNE, 8-OHdG, and COX-2 was performed 24 hours after ischemia. Results: The cilostazol group and the combination therapy group showed significant decreases of infarct volume and significant improvements of rCBF during ischemia, compared with the vehicle or aspirin group. Significant decreases of Bax, TUNEL, 8-OHdG, and 4-HNE expression in the combination therapy group, compared with those in the vehicle or aspirin group, were shown in the boundary zone. COX-2 expression was unexpectedly increased in the combination therapy group. Discussion: Aspirin co-administration did not inhibit this effect. The addition of the oral administration of cilostazol either alone or with aspirin administration may be beneficial for subsequent cerebral ischemic damage in terms of reducing infarct volume, improving rCBF during ischemia, inhibiting the apoptotic pathway, and reducing oxidative stress.

In vivo monitoring of arterially transplanted bone marrow mononuclear cells in a rat transient focal brain ischemia model using magnetic resonance imaging

Abstract Objectives: Intra-arterial transplantation of bone marrow mononuclear cells (BMMCs) effectively improves neuronal function and limits the infarct size. We monitored the fate of BMMCs labeled with super paramagnetic iron oxide (SPIO) until 7 days after the transplantation using high-field magnetic resonance imaging (MRI). Methods: Male Sprague–Dawley rats were subjected to 90-minute focal ischemia using the intraluminal suture technique followed by transplantation of 1×107 BMMCs or vehicle only via the ipsilateral carotid artery immediately after reperfusion. Autologous BMMCs were labeled with SPIO by electroporation prior to ischemia. MRI studies were performed at 1 hour, 24 hours, 3 days, and 7 days after reperfusion on each rat. The total infarct volume and the volume of negative dots were measured on T2-weighted images and T2*-weighted images, respectively. Results: One hour after BMMC transplantation, we confirmed wide spread distribution of BMMCs in the ischemic hemisphere as a negative dot. The volume of negative dot normalized by hemispheric volume decreased rapidly and was seldom seen at the seventh day after transplantation. The infarct volume was significantly smaller in the transplanted group than the vehicle group at 24 hours and 7 days after reperfusion. Discussion: The present study established in vivo monitoring of intra-arterial transplanted SPIO-labeled BMMCs immediately after reperfusion using MRI of a rat transient focal ischemia model. The accumulation of BMMCs in ischemic lesion at the acute stage of ischemia can be part of the conditions to limit the infarct size.

Neuroprotective effects of pretreatment with macrolide antibiotics on cerebral ischemia reperfusion injury

Abstract Objective: This study aims to determine if macrolide antibiotics have neuroprotective effects against transient cerebral ischemia. Methods: Sprague-Dawley rats were subjected to cerebral ischemia for 90 minutes followed by 24 or 72 hours of reperfusion. An oral suspension of roxithromycin (RXM), clarithromycin (CAM), erythromycin (EM), azithromycin (AZM), or kitasamycin (INN) was given at 10 or 100 mg/kg for 7 days before ischemia. The infarct volume, edema volume, and neurological performance were evaluated after 24 and 72 hours of reperfusion. The cerebral blood flow (CBF) was measured with a magnetic resonance imaging (MRI) system after 90 minutes of ischemia. Another experiment was conducted to investigate how the ischemic injury was affected by the interval from the antibiotic pretreatment to the ischemia in rats pretreated with CAM. Results: Roxithromycin, CAM, AZM, and INN significantly reduced the infarct volume in the high-dose group after 24 and 72 hours of reperfusion. All of the agents significantly decreased the edema in the high-dose groups at 24 and 72 hours, while only CAM and AZM significantly reduced the edema volume in the low-dose groups at 24 hours. All of the macrolide antibiotics at the high dose significantly improved neurological deficit scores at 24 and 72 hours. There were no differences in the CBF between the vehicle and respective antibiotic groups. In the experiment examining the interval, the 24-hour interval group exhibited the strongest neuroprotective effect. Discussion: These results demonstrate that the macrolide antibiotics RXM, CAM, EM, AZM, and INN may confer neuroprotective effects against ischemic damage following cerebral ischemia without affecting the CBF.