Neuroprotective effect of nobiletin on cerebral ischemia-reperfusion injury in transient middle cerebral artery-occluded rats.

Nobiletin, a citrus polymethoxylated flavone, is reported to possess various pharmacological activities such as anticancer, anti-inflammation, and antioxidant effects. Recently, nobiletin was shown to provide therapeutic benefit for the treatment of Alzheimer׳s disease by activating cAMP-response element-binding protein (CREB). In the present study, we investigated whether nobiletin could protect the brain against ischemia-reperfusion (I/R) injury and improve functional outcome in cerebral I/R model rats, since CREB activation is known to protect neuronal cells in cerebral ischemia. Nobiletin was injected twice at 0 and 1h after the start of reperfusion in transient middle cerebral artery occlusion (t-MCAO) rats. Cerebral I/R induced prominent brain damage in the ischemic hemisphere of t-MCAO rat brains; however, nobiletin treatment significantly reduced the infarct volume and suppressed the brain edema. Immunohistochemical and TUNEL staining indicated that nobiletin treatment significantly suppressed neutrophil invasion into the ischemic region and significantly decreased apoptotic brain cell death in ischemic hemisphere, suggesting that the anti-inflammatory effect and anti-apoptotic effect should be regarded as the neuroprotective mechanism of nobiletin. Moreover, nobiletin treatment ameliorated motor functional deficits in the ischemic rats compared with those deficits of the vehicle-treated group. These results indicate that nobiletin is a potential neuroprotectant for the treatment of cerebral I/R injury.

Nanoparticles accumulate in ischemic core and penumbra region even when cerebral perfusion is reduced.

The use of a liposomal drug delivery system is a promising strategy for avoiding side effects and enhancing drug efficiency by changing the distribution of the intact drug. We have previously shown that liposomal agents quickly accumulated in an ischemia-reperfusion region and ameliorated cerebral ischemia-reperfusion injury when they were injected after reperfusion in transient middle cerebral artery occlusion (t-MCAO) rats. In the present study, we hypothesized that liposomes also act effectively as a drug carrier in the ischemic state, since the integrity of the blood brain barrier is disrupted at an early stage after an ischemic event. To test this hypothesis, the cerebral distribution of fluorescence-labeled liposomes was observed in permanent MCAO (p-MCAO) rats. The liposomes accumulated in the ischemic core and the penumbra region when injected at 1 or 2h after occlusion. The accumulation in the ischemic core region was clearly greater than that in the penumbra region, despite the cerebral blood perfusion of the core region being substantially reduced. This result suggests that drug delivery to an ischemic region using liposomes is possible even when cerebral blood circulation has not recovered. Because liposomal drug delivery systems have the potential to effectively employ a number of agents that have failed in clinical trials, they may offer an effective strategy for achieving neuroprotection in stroke patients.

Treatment of cerebral ischemia-reperfusion injury with PEGylated liposomes encapsulating FK506.

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.

A single injection of liposomal asialo-erythropoietin improves motor function deficit caused by cerebral ischemia/reperfusion.

Modification of the liposomal surface with a targeting molecule is a promising approach for the targeted delivery of therapeutics. Asialo-erythropoietin (AEPO) is a potent tool for targeting an ischemic region by binding to the EPO receptors on neuronal cells. Additionally, it shows a strong cytoprotective effect against programed cell death. Hence, AEPO-modified liposomes appear likely to have both a neuronal-targeting character and a neuroprotective effect on cerebral ischemic injury. In this study, we assessed the targeting ability of AEPO-modified PEGylated liposomes (AEPO-liposomes) to ischemic region and their improvement effect on neurological deficits induced by ischemia/reperfusion (I/R) in transient middle cerebral artery occlusion (t-MCAO) rats. Immunohistological analysis showed that the AEPO-liposomes given immediately after reperfusion extravasated into the ischemic region and attached strongly to neuronal cells. Also, neuronal nuclei (NeuN) staining was clearly visible only in the AEPO-liposome-treated group, suggesting that AEPO-liposomes protected neuronal cells from ischemia/reperfusion-induced damage. Moreover, a single administration of low-dose AEPO-liposomes significantly improved the neurological deficit compared to vehicle and free-AEPO treatment at 7 days after injection. In conclusion, AEPO-liposomes have clear potential as a neuroprotectant after stroke and as a DDS device targeting ischemic regions.

Amelioration of cerebral ischemia-reperfusion injury based on liposomal drug delivery system with asialo-erythropoietin.

Cerebral ischemia-reperfusion (I/R) injury induces secondary cerebral damage. As drugs for treating this type of injury have shown poor efficacy and adverse side effects in clinical trials, a novel therapeutic strategy has been long awaited. In this study, we focused on the disruption of the blood-brain barrier after stroke, and applied a liposomal drug delivery system (DDS) designed to enhance the pharmacological effect of the neuroprotectant and to avoid side effects. PEGylated liposomes were injected at varying time after the start of reperfusion in transient middle cerebral artery occlusion (t-MCAO) model rats. The results showed PEGylated liposomes accumulated in the ischemic hemisphere at an early stage after reperfusion and were retained in the lesion for at least 24h after injection. We also investigated the effectiveness of asialo-erythropoietin (AEPO)-modified PEGylated liposomes (AEPO-liposomes) in treating the cerebral I/R injury. AEPO-liposome treatment significantly reduced TTC-defined cerebral legion following cerebral I/R injury, and ameliorated motor function compared with vehicle and AEPO treatment. In conclusion, these results indicate that AEPO-liposomes are a promising liposomal formulation for protecting the brain from I/R injury, and that this liposomal DDS has potential as a novel strategy for the treatment of cerebral I/R injury.