Melatonin receptor agonist ramelteon attenuates mouse acute and chronic ischemic brain injury.

Melatonin receptors (MTs) are potential drug targets for stroke therapy. Ramelteon is a selective melatonin receptor agonist used to treat insomnia. In this study we investigated whether ramelteon could attenuate cerebral ischemia in mice. Acute focal cerebral ischemia was induced in mice via middle cerebral artery occlusion (MCAO). We found oral administration of ramelteon (3.0 mg/kg) significantly attenuated ischemic injury even when it was given 4 h after the onset of ischemia. We showed that administration of ramelteon (3.0 mg/kg) displayed comparable protective efficacy and length of effective time window as administration of edaravone (10 mg/kg, i.p.), which was used in clinic to treat ischemic stroke. Chronic ischemic brain injury was induced in mice using photothrombosis. Oral administration of ramelteon (3.0 mg · kg-1 · d-1) for 7 days after ischemia significantly attenuated functional deficits for at least 15 days. The neuroprotection of ramelteon was blocked by 4-P-PDOT, a specific MT antagonist. We further revealed that ramelteon significantly inhibited autophagy in the peri-infarct cortex in both the mouse ischemia models via regulating AMPK/mTOR signaling pathway. Intracerebroventricular injection of rapamycin, an autophagy activator, compromised the neuroprotection of ramelteon, suggesting ramelteon might attenuate ischemic injury by counteracting autophagic cell death. These data demonstrate for the first time the potential benefits of ramelteon in the treatment of both acute and chronic ischemic brain injury and provide the rationale for the application of ramelteon in stroke therapy.

Urolithin A-activated autophagy but not mitophagy protects against ischemic neuronal injury by inhibiting ER stress in vitro and in vivo.

AIM: Mitochondrial autophagy (mitophagy) clears damaged mitochondria and attenuates ischemic neuronal injury. Urolithin A (Uro-A) activates mitophagy in mammal cells and Caenorhabditis elegans. We explored neuroprotection of Uro-A against ischemic neuronal injury. METHODS: Mice were subjected to middle cerebral artery occlusion. The brain infarct and neurological deficit scores were measured. The N2a cells and primary cultured mice cortical neurons were subjected to oxygen-glucose deprivation and reperfusion (OGD/R). Uro-A was incubated during OGD/R, and cell injury was determined by MTT and LDH. Autophagosomes were visualized by transfecting mCherry-microtubule-associated protein 1 light chain 3 (LC3). The protein levels of LC3-II, p62, Translocase Of Inner Mitochondrial Membrane 23 (TIMM23), and cytochrome c oxidase subunit 4 isoform 1 (COX4I1) were detected by Western blot. The ER stress markers, activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), were determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Urolithin A alleviated OGD/R-induced injury in N2a cells and neurons and reduced ischemic brain injury in mice. Uro-A reinforced ischemia-induced autophagy. Furthermore, Uro-A-conferred protection was abolished by 3-methyladenine, suggesting the requirement of autophagy for neuroprotection. However, mitophagy was not further activated by Uro-A. Instead, Uro-A attenuated OGD/R-induced ER stress, which was abolished by 3-methyladenosine. Additionally, neuroprotection was reversed by ER stress inducer. CONCLUSION: Urolithin A protected against ischemic neuronal injury by reinforcing autophagy rather than mitophagy. Autophagy activation by Uro-A attenuated ischemic neuronal death by suppressing ER stress.

Mitochondrial transport serves as a mitochondrial quality control strategy in axons: Implications for central nervous system disorders.

Axonal mitochondrial quality is essential for neuronal health and functions. Compromised mitochondrial quality, reflected by loss of membrane potential, collapse of ATP production, abnormal morphology, burst of reactive oxygen species generation, and impaired Ca2+ buffering capacity, can alter mitochondrial transport. Mitochondrial transport in turn maintains axonal mitochondrial homeostasis in several ways. Newly generated mitochondria are anterogradely transported along with axon from soma to replenish axonal mitochondrial pool, while damaged mitochondria undergo retrograde transport for repair or degradation. Besides, mitochondria are also arrested in axon to quarantine damages locally. Accumulating evidence suggests abnormal mitochondrial transport leads to mitochondrial dysfunction and axon degeneration in a variety of neurological and psychiatric disorders. Further investigations into the details of this process would help to extend our understanding of various neurological diseases and shed light on the corresponding therapies.

Pre-stroke Metformin Treatment is Neuroprotective Involving AMPK Reduction.

Long-term metformin treatment reduces the risk of stroke. However, the effective administration pattern and indications of metformin on acute cerebral ischemia are unclear. To investigate the neuroprotective treatment duration and dosage of metformin on focal ischemia mice and the association of neuroprotection with 5'-adenosine monophosphate-activated protein kinase (AMPK) regulations, male C57BL/6 mice were subjected to permanent or transient middle cerebral artery occlusion (MCAO) and metformin of 3, 10 and 30 mg/kg was intraperitoneally injected 1, 3 or 7 days prior to MCAO, or at the onset, or 1, 3 or 6 h after reperfusion, respectively. Infarct volumes, neurological deficit score, cell apoptosis, both total and phosphorylated AMPK expressions were assessed. Results showed that prolonged pretreatment to 7 days of metformin (10 mg/kg) significantly ameliorated brain infarct, neurological scores and cell apoptosis in permanent MCAO mice. Shorter (3 days or 1 day) or without pretreatment of metformin was not effective, suggesting a pretreatment time window. In transient MCAO mice, metformin showed no neuroprotection even with pretreatment. The expressions of total and phosphorylated AMPK were sharply decreased with effective metformin pretreatments in ischemic brains. Our data provided the first evidence that in acute ischemic injury, a 7-days pretreatment duration of 10 mg/kg metformin is necessary for its neuroprotection, and metformin may not be beneficial in the cases of blood reperfusion.

Electrophoretic deposition to promote layer-by-layer assembly for in situ gene delivery application.

In this study, layer-by-layer (LbL) assembly of polyelectrolyte was facilitated using electric field assistance (EFA). To elucidate the EFA effects on polyelectrolyte multilayers (PEMs), the electric fields were solely administrated to either chitosan or DNA adsorption. Both DNA and chitosan adsorptions can be augmented under low electric field due to the electrophoretic deposition. However, the ensuing electrochemical reactions on electrode interfered with interactions between multilayers when the bias was larger than 0.5V, leading to retard deposition. Subsequent delivery experiments indicated that EFA during DNA deposition demonstrated superior DNA release. In contrast, no obvious improvements were observed for the groups using the EFA during chitosan deposition. Moreover, water contact angle experiments revealed different effects of electric fields on multilayer structure. Using EFA during DNA deposition led to a layered-form composition, whereas interpenetration of electrolytes was enhanced with the application of the electric field during chitosan deposition. For in vitro experiments, EFA during DNA deposition significantly enhanced in situ transfection performances of PEMs that the transgene expression levels were increased and the periods were extended, suggesting this method is potential to quantitatively and temporally improve substrate-mediated gene delivery.

Inhibition of G protein-coupled receptor 81 (GPR81) protects against ischemic brain injury.

AIM: Lactates accumulate in ischemic brains. G protein-coupled receptor 81 (GPR81) is an endogenous receptor for lactate. We aimed to explore whether lactate is involved in ischemic injury via activating GPR81. METHODS: N2A cells were transfected with GFP-GPR81 plasmids 24 h previously, and then treated with GPR81 antagonist 3-hydroxy-butyrate (3-OBA) alone or cotreated with agonists lactate or 3, 5-dihydroxybenzoic acid (3, 5-DHBA) during 3 h of oxygen-glucose deprivation (OGD). Adult male C57BL/6J mice and primary cultured cortical neurons were treated with 3-OBA at the onset of middle cerebral artery occlusion (MCAO) or OGD, respectively. RESULTS: The GPR81 overexpression increased the cell vulnerability to ischemic injury. And GPR81 antagonism by 3-OBA significantly prevented cell death and brain injury after OGD and MCAO, respectively. Furthermore, inhibition of GPR81 reversed ischemia-induced apoptosis and extracellular signal-regulated kinase (ERK) signaling may be involved in the neuroprotection. CONCLUSIONS: G protein-coupled receptor 81 (GPR81) inhibition attenuated ischemic neuronal death. Lactate may aggravate ischemic brain injury by activating GPR81. GPR81 antagonism might be a novel therapeutic strategy for the treatment of cerebral ischemia.