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Aims Rats subjected to transient focal ischemia and cultured neuronal cells subjected to oxygen-glucose deprivation (OGD) were treated with clarithromycin (CAM) to evaluate the effects of CAM in protecting against neuronal damage. Main methods Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min and then reperfused. Each animal was given an oral dose clarithromycin (CAM, 100 mg/kg) or vehicle alone just after the ischemia was commenced. The infarct volume, edema index and neurological performance were assessed after 24 and 72 h of reperfusion. The cerebral blood flow (CBF) was measured with an MRI system at 90 min after MCAO. After 24 and 72 h, oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) were assessed by immunohistochemical analyses and degenerative cells were assessed in the cortex by Fluoro-Jade C (FJC) labeling. The cultured neuronal cells were also used to examine the effects of CAM exposure on the viability of the cells after OGD. Key findings CBF was unchanged between the two groups. Significant reductions of the infarct volume and edema index, an improved neurological deficit score, a significant suppression of 4-HNE and 8-OHdG expression, marked reductions of Iba-1 and TNF-α expression, and a significant reduction of FJC-positive cells were also observed in the CAM-treated animals at both time points. Treatment with 10 μM and 100 μM CAM in vitro significantly reduced cell death after OGD. Significance CAM appears to provide antioxidant and anti-inflammatory effects and protect against neuronal damage after cerebral ischemia and OGD. [ABSTRACT FROM AUTHOR]

Objective: This study aims to determine if erythromycin provides neuroprotective effects against ischemic injury following permanent focal cerebral ischemia. Methods: Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). Each animal received a single subcutaneous injection of erythromycin lactobionate (EM, 50 mg/kg) or vehicle immediately after ischemia. The infarct volume, edema index and neurological performance were evaluated at 24 and 72 h after MCAO. The cerebral blood flow (CBF) was measured with an MRI system at 30 min after MCAO. TUNEL staining and immunohistochemical analyses for oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) in the cortex were conducted at 24 and 72 h after MCAO. Results: The CBF did not differ between the EM-treated and vehicle-treated groups. The EM treatment significantly reduced the infarct volume (p < 0.01) at 24 and 72 h after MCAO and significantly reduced the edema index (p < 0.01) at 24 h. The EM treatment significantly improved the neurological deficit scores (p < 0.05) at 24 and 72 h. EM also 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 < 0.05) at both time points. The EM treatment significantly reduced TUNEL-positive cells (p < 0.01) at both time points. Conclusion: These findings suggest that EM can protect against the neuronal damage caused by cerebral ischemia by alleviating inflammation and reducing oxidant stress. [ABSTRACT FROM AUTHOR]

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 highdose 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 x 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. [ABSTRACT FROM AUTHOR]

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.Journal of Cerebral Blood Flow & Metabolism (2009) 29, 715–725; doi:10.1038/jcbfm.2008.164; published online 14 January 2009 [ABSTRACT FROM AUTHOR]

Abstract: Aims: Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window. Main methods: Male Sprague–Dawley rats were subjected to 90minute focal cerebral ischemia, followed by intravenous administration of 1×107 allogeneic BMMCs or vehicle at 0, 3 or 6h after reperfusion or 2×107 BMMCs 6h after reperfusion. Other rats administered 1×107 BMMCs at 6h after reperfusion received additional BMMC transplantation or vehicle 9h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72h after reperfusion. Key findings: Infarct volumes at 24h were significantly decreased in transplantation rats at 0 and 3h, but not at 6h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine expression at 24 and 72h after reperfusion. Significance: Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was >3h and <6h after reperfusion in this model. Repeated transplantation at 6 and 9h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection. [Copyright &y& Elsevier]

Abstract: 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 7days before MCAO. In the post-treatment, rats received atorvastatin or vehicle for 7days after MCAO. Measurement of infarct volume, as well as neurological and immunohistochemical assessments, were done 24h and 7days 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 7days 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 7days 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. [Copyright &y& Elsevier]

Abstract: 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 90min 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. [Copyright &y& Elsevier]

Many drugs for cerebral infarction that were shown to be effective in animal experiments have shown negative results in human clinical trials. For this reason, a completely new approach is needed to develop brain protection therapies against cerebral infarction. Brain protection therapies can be categorized into 3 types: 1) lengthening the therapeutic time window for thrombolytic therapy, 2) reducing the side effects of thrombolytic therapy, and 3) brain protection drug therapy for patients with contraindications for thrombolytic therapy (including combination therapy). Here, we show our recent results of brain protection therapy. First, combination therapy with 2 effective drugs was tried, and time-lag administration was performed. Combination therapy was effective and lengthened the therapeutic time window. Next, a completely new approach to improve cerebral ischemic damage, namely, H2 gas inhalation therapy, was tried. This therapy was also effective, even in the ischemic core. [ABSTRACT FROM AUTHOR]

Abstract: We previously reported that the protein transduction domain fused FNK (PTD-FNK) protein, which was derived from anti-apoptotic Bcl-xL protein and thereby gained higher anti-cell death activity, has a strong neuroprotective effect on rat focal brain ischemia models. The aim of this study was to investigate the effect of PTD-FNK protein and hypothermia combined therapy on cerebral infarction. Male SD rats were subjected to 120min middle cerebral artery occlusion (MCAO) with intraluminal thread. Rats were divided into 4 groups: 1) 37°C vehicle administration (37V); 2) 37°C PTD-FNK administration (37F); 3) 35°C vehicle administration (35V); and 4) 35°C PTD-FNK administration (35F). PTD-FNK protein was intravenously administered 60min after the induction of MCAO. Hypothermia (35°C) was applied during 120min MCAO. Rats were sacrificed 24h later; infarct volumes were measured, and Bax, Bcl-2, TUNEL and caspase-12 immunostaining was evaluated. There was significant infarct volume reduction in 37F, 35V, and 35F groups compared to 37V. There was also a significant difference between 37F and 35F. This suggests that hypothermia enhanced the effect of PTD-FNK. Similar results were found in neurological symptoms. Caspase-12 and TUNEL staining showed a significant difference between 37F and 35F; however, Bax and Bcl-2 staining failed to show a difference. In this study we showed an additive protective effect of hypothermia on PTD-FNK treatment, and immunohistological results showed that the protective mechanisms might involve the inhibition of apoptotic pathways through caspase-12, but not through Bcl-2. [Copyright &y& Elsevier]

Abstract: 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 90min 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 1day after combination therapy compared with monotherapy, and this neuroprotection continued for 7days. 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. [Copyright &y& Elsevier]

Abstract: Little work has been performed to determine roles of mitochondrial ATP-sensitive potassium channels (mitoKATP) in ischemic preconditioning (IPC) in brain. To investigate the role on cerebral IPC, we examined effect of 5-hydroxydecanoate (5-HD), a selective mitoKATP blocker, and diazoxide (DZX), a selective mitoKATP opener on various IPC models. An IPC model with gerbil: 2 min bilateral common carotid arteries occlusion (BLCO)+24 h recovery+5 min BLCO. 5-HD, DZX, vehicle was administered 30 min before 5 min BLCO. Seven days later, surviving CA1 neurons were counted. A focal IPC model with rat: 15 min middle cerebral artery occlusion (MCAO)+48 h recovery+90 min MCAO. Twenty-four hours before 90 min MCAO, 5-HD, DZX, or vehicle was administered. One day after 90 min MCAO, neurological symptoms and infarct volumes were evaluated. An in vitro IPC model with primary neuronal cultures: 8 min oxygen–glucose deprivation (OGD)+24 h recovery+70 min OGD. Thirty minutes before 70 min OGD, 5-HD or DZX were added. One day later, surviving neurons were counted. Mitochondrial membrane potential was also monitored. 5-HD significantly attenuated the protective effect of IPC in gerbil model, rat model, and in vitro OGD model. DZX significantly facilitated the protective effect of IPC in gerbil and rat model. The mitochondrial membranes were depolarized with IPC, and 5-HD treatment significantly reduced this effect. These results strongly suggest that mitoKATP channel activation plays a key role in development of a protective mechanism of cerebral IPC. [Copyright &y& Elsevier]

Abstract: 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 1×107 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. [Copyright &y& Elsevier]

To clarify regional difference in 1H magnetic resonance spectral changes in the acute stage of focal cerebral ischemia, and to assess whether any correlation exists between spectral changes in acute stage and pathological outcome in the chronic stage, we measured the non-volume selected 1H-MRSI serially in the acute stage of focal ischemia in rat brain and correlated histopathological findings. The focal ischemia was induced by intraluminal MCA thread occlusion. After induction of focal ischemia, 1H-MR Spectroscopic Images (MRSI) were taken serially up to 10 h after the occlusion. NAA in the peri-ischemic area was maintained. On the other hand, the decline of NAA in the ischemic lesion was linear and drastic during the first 10 h after MCA occlusion, and the rates of decline of NAA concentrations up to 10 h after induction of ischemia, calculated with linear regression, were 0.44 m mol/kg/h in the ischemic core and 0.29 m mol/kg/h in the ischemic rim (p<0.05). Lactate accumulated significantly in all three areas. Decreasing depth of NAA during the acute stage of cerebral ischemia identifies the formation of the ischemic core and can be used as a predictor for histological outcome. [Copyright &y& Elsevier]

rt-PA improves neurological outcome if it is administered at early time of stroke, and intracerebral hemorrhage is the main complication of thrombolytic therapy and it proves sometimes fatal. The aims of this study are to clarify the correlation between the efficacy of rt-PA, the severity of hemorrhage and treatment delay, using the developed embolic infarction model. Fibrin-rich clots were used to occlude MCA on Sprague–Dawley rats. Alteplase of 10 mg/kg was administrated via femoral vein at 30 (n=7), 60 (n=10), 90 (n=6) and 120 (n=6) min after clot embolization and vehicle group (n=10) received saline. Twenty four hours after embolization, brains were perfused with PBS and TTC stain was utilized to measure the infarct volume. Hemorrhage volume was quantified with spectrophotometric assay of extraction from ischemic hemisphere. Infarction was significantly smaller in the groups administrated altepalse at 30, 60 and 90 min and hemorrhage was significantly less in the group of 30 min than vehicle group. However, administration of alteplase at 120 min did not ameliorate infarction and more significantly increased hemorrhage than vehicle group. In these results, we clearly showed that vascular therapeutic time window of this model can be within 120 min and treatment delay of thrombolysis enhanced the severity of hemorrhagic transformation. [Copyright &y& Elsevier]

NS-7 is a novel, voltage-dependent Na+ and Ca2+ channel blocker. This study evaluated the in vivo neuroprotective effect of NS-7 in a rat transient focal ischemic model when administered during occlusion. Left middle cerebral artery occlusion was induced in adult male Sprague–Dawley rats for 120 min using an intraluminal thread method. The rats received a single intravenous injection of NS-7 or saline (control group) just after the onset of ischemia, and at 30, 60 and 120 min after ischemia. Their brains were removed after 48 h reperfusion, sectioned, and stained with hematoxylin and eosin. Animals were evaluated by neurological examination at 120 min ischemia and 48 h reperfusion. Infarcted cortex and striatum were measured quantitatively and infarction volumes were calculated. Cortical infarction volumes were 128±74 (NS-7) and 214±64 mm3 (control) immediately after the ischemia group, 155±48 (NS-7) and 225±12 mm3 (control) after the 30 min group, 160±54 (NS-7) and 225±48 mm3 (control) after the 60 min group, and 176±43 (NS-7) and 223±38 mm3 (control) after the 120 min group. Cortices in NS-7-treated groups were significantly less infarcted than in control groups at all treatment times. There was no significant difference in the striatal infarction volume between the treatment and control groups. Neurological examination showed that hemiparesis and abnormal posture of the NS-7 groups were significantly more improved at 48 h reperfusion than those of the control groups without posture examination in the 120 min group. These observations suggest that NS-7 may be a new potential therapeutic agent for the acute phase of cerebral infarction. [Copyright &y& Elsevier]