sirolimus and Brain-Ischemia

Ischemic stroke remains a leading cause of morbidity and disability around the world. The sequelae of serious neurological damage are irreversible due to body's own limited repair capacity. However, endogenous neurogenesis induced by cerebral ischemia plays a critical role in the repair and regeneration of impaired neural cells after ischemic brain injury. mTOR (mammalian target of rapamycin) kinase has been suggested to regulate neural stem cells ability to self-renew and differentiate into proliferative daughter cells, thus leading to improved cell growth, proliferation, and survival. In this review, we summarized the current evidence to support that mTOR signaling pathways may enhance neurogenesis, angiogenesis, and synaptic plasticity following cerebral ischemia, which could highlight the potential of mTOR to be a viable therapeutic target for the treatment of ischemic brain injury.

Topics: Brain Injuries; Brain Ischemia; Cerebral Infarction; Humans; Neurogenesis; Sirolimus; TOR Serine-Threonine Kinases

The significant morbidity that accompanies stroke makes it one of the world's most devastating neurological disorders. Currently, proven effective therapies have been limited to thrombolysis and thrombectomy. The window for the administration of these therapies is narrow, hampered by the necessity of rapidly imaging patients. A therapy that could extend this window by protecting neurons may improve outcome. Endogenous neuroprotection has been shown to be, in part, due to changes in mTOR signalling pathways and the instigation of productive autophagy. Inducing this effect pharmacologically could improve clinical outcomes. One such therapy already in use in transplant medicine is the mTOR inhibitor rapamycin. Recent evidence suggests that rapamycin is neuroprotective, not only via neuronal autophagy but also through its broader effects on other cells of the neurovascular unit. This review highlights the potential use of rapamycin as a multimodal therapy, acting on the blood-brain barrier, cerebral blood flow and inflammation, as well as directly on neurons. There is significant potential in applying this old drug in new ways to improve functional outcomes for patients after stroke.

Topics: Anti-Bacterial Agents; Brain Ischemia; Drug Repositioning; Humans; Neuroprotective Agents; Sirolimus; Stroke; TOR Serine-Threonine Kinases; Treatment Outcome

Amplifying endogenous neuroprotective mechanisms is a promising avenue for stroke therapy. One target is mammalian target of rapamycin (mTOR), a serine/threonine kinase regulating cell proliferation, cell survival, protein synthesis, and autophagy. Animal studies investigating the effect of rapamycin on mTOR inhibition following cerebral ischemia have shown conflicting results.. To conduct a systematic review and meta-analysis evaluating the effectiveness of rapamycin in reducing infarct volume in animal models of ischemic stroke.. Our search identified 328 publications. Seventeen publications met inclusion criteria (52 comparisons: 30 reported infarct size and 22 reported neurobehavioral score). Study quality was modest (median 4 of 9) with no evidence of publication bias. The point estimate for the effect of rapamycin was a 21.6% (95% CI, 7.6%-35.7% p < 0.01) improvement in infarct volume and 30.5% (95% CI 17.2%-43.8%, p < 0.0001) improvement in neuroscores. Effect sizes were greatest in studies using lower doses of rapamycin.. Low-dose rapamycin treatment may be an effective therapeutic option for stroke. Modest study quality means there is a potential risk of bias. We recommend further high-quality preclinical studies on rapamycin in stroke before progressing to clinical trials.

Topics: Animals; Brain Ischemia; Cerebellum; Disease Models, Animal; Humans; Immunosuppressive Agents; Neuroprotective Agents; Sirolimus; Stroke; TOR Serine-Threonine Kinases

Given the current limitation of therapeutic approach for ischemic stroke, a leading cause of disability and mortality in the developed countries, to develop new therapeutic strategies for this devastating disease is urgently necessary. As a serine/threonine kinase, mammalian target of rapamycin (mTOR) activation can mediate broad biological activities that include protein synthesis, cytoskeleton organization, and cell survival. mTOR functions through mTORC1 and mTORC2 complexes and their multiple downstream substrates, such as eukaryotic initiation factor 4E-binding protein 1, p70 ribosomal S6 kinase, sterol regulatory element-binding protein 1, hypoxia inducible factor-1, and signal transducer and activator transcription 3, Yin Ying 1, Akt, protein kinase c-alpha, Rho GTPase, serum-and gucocorticoid-induced protein kinase 1, etc. Specially, the role of mTOR in the central nervous system has been attracting considerable attention. Based on the ability of mTOR to prevent neuronal apoptosis, inhibit autophagic cell death, promote neurogenesis, and improve angiogenesis, mTOR may acquire the capability of limiting the ischemic neuronal death and promoting the neurological recovery. Consequently, to regulate the activity of mTOR holds a potential as a novel therapeutic strategy for ischemic stroke.

Topics: Animals; Brain; Brain Infarction; Brain Ischemia; Humans; Neovascularization, Physiologic; Neurogenesis; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The Syntax score (SXscore) was recently developed as a comprehensive angiographic scoring system aiming to assist in patient selection and risk stratification of patients with extensive coronary artery disease undergoing contemporary revascularization. A validation of this angiographic classification scheme is lacking. We assessed its predictive value in patients who underwent percutaneous intervention (PCI) for 3-vessel disease and explored its performance in comparison with the modified lesion classification system of the American Heart Association/American College of Cardiology. The SXscore, applied to 1,292 lesions in 306 patients who underwent PCI for 3-vessel disease in the Arterial Revascularization Therapies Study Part II, was 4 to 54.5, and after a median of 370 days (range 274 to 400) predicted the rate of major adverse cardiac and cerebrovascular events (hazard ratio 1.08/U increase, 95% confidence interval 1.05 to 1.11, p <0.0001), with patients in the highest SXscore tertile having a significantly higher event rate (27.9%) than patients in the lowest tertile (8.7%, hazard ratio 3.5, 95% confidence interval 1.7 to 7.4, p = 0.001). By multivariable analyses, SXscore independently predicted outcome with an almost fourfold adjusted increase in the risk of major adverse cardiac and cerebrovascular events in patients with high versus low values based on the discrimination level provided by classification and regression tree analysis. Compared with the modified lesion classification scheme of the American Heart Association/American College of Cardiology, SXscore showed a greater discrimination ability (c-index 0.58 +/- 0.08 vs 0.67 +/- 0.08, respectively, p <0.001) and a better goodness of fit with the Hosmer-Lemeshow statistic. In conclusion, the SXscore is a promising tool to risk stratify outcome in patients with extensive coronary artery disease undergoing contemporary PCI.

Topics: Aged; Angioplasty, Balloon, Coronary; Brain Ischemia; Coronary Angiography; Coronary Disease; Female; Fibrinolytic Agents; Follow-Up Studies; Forecasting; Humans; Ischemic Attack, Transient; Longitudinal Studies; Male; Middle Aged; Myocardial Infarction; Platelet Aggregation Inhibitors; Risk Assessment; Sirolimus; Stents; Stroke; Survival Rate; Treatment Outcome

To investigate the effect of rapamycin on mitochondrial dynamic balance in diabetic rats subjected to cerebral ischemia-reperfusion injury. Male Sprague Dawley (SD) rats (n = 78) were treated with high fat diet combined with streptozotocin injection to construct diabetic model in rats. Transient middle cerebral artery occlusion (MCAO) of 2 hours was induced and the brains were harvested after 1 and 3 days of reperfusion. Rapamycin was injected intraperitoneally for 3 days prior to and immediately after operation, once a day. The neurological function was assessed, infarct volumes were measured and HE staining as well as immunohistochemistry were performed. The protein of hippocampus was extracted and Western blotting were performed to detect the levels of mTOR, mitochondrial dynamin related proteins (DRP1, p-DRP1, OPA1), SIRT3, and Nix/BNIP3L. Diabetic hyperglycemia worsened the neurological function performance (p < 0.01), enlarged infarct size (p < 0.01) and increased ischemic neuronal cell death (p < 0.01). The increased damage was associated with elevations of p-mTOR, p-S6, and p-DRP1; and suppressions of SIRT3 and Nix/BNIP3L. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and reversed the biomarker alterations caused by diabetes. High glucose activated mTOR pathway and caused mitochondrial dynamics toward fission. The protective effect of rapamycin against diabetes-enhanced ischemic brain damage was associated with inhibiting mTOR pathway, redressing mitochondrial dynamic imbalance, and elevating SIRT3 and Nix/BNIP3L expression.

Topics: Animals; Apoptosis Regulatory Proteins; Brain; Brain Injuries; Brain Ischemia; Diabetes Mellitus, Experimental; Infarction, Middle Cerebral Artery; Male; Mitochondrial Dynamics; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Sirtuin 3; TOR Serine-Threonine Kinases

Electroacupuncture (EA) pretreatment has been shown to alleviate cerebral ischemia-reperfusion (I/R) injury; however, the underlying mechanism remains unclear. To investigate the involvement of mTOR signaling in the protective role of EA in I/R-induced brain damage and mitochondrial injury.. Sprague-Dawley male rats were pretreated with vehicle, EA (at Baihui and Shuigou acupoints), or rapamycin + EA for 30 min daily for 5 consecutive days, followed by the middle cerebral artery occlusion to induce I/R injury. The neurological functions of the rats were assessed using the Longa neurological deficit scores. The rats were sacrificed immediately after neurological function assessment. The brains were obtained for the measurements of cerebral infarct area. The mitochondrial structural alterations were observed under transmission electron microscopy. The mitochondrial membrane potential changes were detected by JC-1 staining. The alterations in autophagy-related protein expression were examined using Western blot analysis.. Compared with untreated I/R rats, EA-pretreated rats exhibited significantly decreased neurological deficit scores and cerebral infarct volumes. EA pretreatment also reversed I/R-induced mitochondrial structural abnormalities and loss of mitochondrial membrane potential. Furthermore, EA pretreatment downregulated the protein expression of LC3-II, p-ULK1, and FUNDC1 while upregulating the protein expression of p-mTORC1 and LC3-I. Rapamycin effectively blocked the above-mentioned effects of EA.. EA pretreatment at Baihui and Shuigou alleviates cerebral I/R injury and mitochondrial impairment in rats through activating the mTORC1 signaling. The suppression of autophagy-related p-ULK1/FUNDC1 pathway is involved in the neuroprotective effects of EA.

Topics: Animals; Autophagy-Related Protein-1 Homolog; Brain Ischemia; Electroacupuncture; Infarction, Middle Cerebral Artery; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mitochondrial Proteins; Mitophagy; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; TOR Serine-Threonine Kinases

Stroke exacts a heavy toll on death and disability worldwide. In animal studies, cell transplant has shown a positive effect by inducing neurogenesis, angiogenesis, and modulating inflammation. Cell transplant therapy could provide researchers with new strategies for treating stroke. The mechanistic target of rapamycin is a central signaling pathway for coordination and control; the administration of rapamycin, a key modulator of this pathway, could be a new therapeutic approach in neurological disorders.. Adult rats were grouped into 5 main groups: control, sham, rapamycin receiving, Sertoli cell receiving, and rapamycin plus Sertoli cell receiving groups. Sertoli cells were taken from another rat tissue and injected into the right striatum region. After 5 days, ischemic induction was performed, and rapamycin injection (300 mg/kg) was performed 1 hour before surgery. After 24 hours, some regions of the brain, including the cortex, striatum, and piriform cortex-amygdala, were isolated for evaluation.. Our results showed that infarct volume, brain edema, and blood-brain barrier permeability assessments were significantly reduced in some areas of the brain in rats that received rapamycin plus Sertoli cells compared with results shown in the control group.. Pretreatment with Sertoli cell transplant plus rapamycin injection may enhance neural survival during ischemia through increased glial cell-derived neurotrophic factor and vascular endothelial growth factor, inhibiting the mechanistic target of rapamycin pathway and increasing autophagy performance.

Topics: Animals; Brain Ischemia; Cell Transplantation; Humans; Infarction, Middle Cerebral Artery; Ischemia; Male; Rats; Sertoli Cells; Sirolimus; Stroke; Treatment Outcome; Vascular Endothelial Growth Factor A

Pre-ischemic hyperglycemia increases the occurrence of post-ischemic seizures both in experimental and clinical settings. The underlying mechanisms are not fully delineated; however, activation of mammalian target of rapamycin (mTOR) has been shown to be engaged in the pathogenesis of epilepsy, in which seizures are a regular occurrence. Therefore, we wanted to explore specifically the capacity of an mTOR inhibitor, rapamycin, in preventing post-ischemic seizures in hyperglycemic rats and to explore the underlying molecular mechanisms. The results showed that none of the rats in the sham control, EG ischemic, or within 3 h of I/R in hyperglycemic ischemic groups experienced seizures. Generalized tonic-clonic seizures were observed in all 8/8 of hyperglycemic ischemic rats at 16 h of I/R. Treatment with rapamycin successfully blocked post-ischemic seizures in 7/8 hyperglycemic ischemic animals. Rapamycin also lessened the neuronal death extraordinarily in hyperglycemic ischemic animals as revealed by histopathological studies. Protein analysis revealed that transient ischemia resulted in increases in p-mTOR and p-S6, especially in the hippocampi of the hyperglycemic ischemic rats. Rapamycin treatment completely blocked mTOR activation. Furthermore, hyperglycemic ischemia induced a much prominent rise of p-ERK1/2 both in the cortex and the hippocampi compared with EG counterparts; whereas rapamycin suppressed it. We conclude that the development of post-ischemic seizures in the hyperglycemic animals may be associated with activations of mTOR and ERK1/2 pathways and that rapamycin treatment inhibited the post-ischemic seizures effectively by suppressing the mTOR and ERK1/2 signaling.

Topics: Animals; Anticonvulsants; Brain Ischemia; Cell Death; Cerebral Cortex; Cytosol; Disease Models, Animal; Hippocampus; Hyperglycemia; Male; MAP Kinase Signaling System; Neurons; Phosphorylation; Rats; Rats, Wistar; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Stroke is one of the leading causes of death in the world, but the underlying molecular mechanism of this disease remains elusive, thus it will be great challenges to finding appropriate protection. MicroRNAs are short, single-stranded, non-coding RNAs and recent studies have shown that they are aberrantly expressed in ischemic condition. Due to the fact that miR-1 has harmful effects on neural damages during brain ischemia, limited miR-1 has been proven to be protective in middle cerebral artery occlusion (MCAO). Here, the possible positive effect of intravenous injection of antagomiR-1 as a post-ischemic treatment on neurological deficits, infarct volume, brain edema and blood-brain barrier (BBB) permeability was evaluated. The rats were divided randomly into three experimental groups, each with 21 animals. MCAO surgery was performed on all groups and one hour later, 0.1 ml normal saline, 0.1 ml rapamycin and 300 pmol/g miR-1 antagomir (soluble in 0.1 ml normal saline), were injected intravenously into control, positive control and treatment group, respectively. After 24 h, neurologic deficits score, infarct volume, brain edema and BBB permeability were measured. The results indicated that post-treatment with miR-1 antagomir significantly improved neurological deficits and reduced infarction volume, brain edema, and BBB permeability. These data proved that there is a positive effects of antagomiR-1 on ischemic neuronal injury and neurological impairment. Due to the fact that microRNAs are able to protect the brain, it would be a promising therapeutic approach to stroke treatment.

Topics: Administration, Intravenous; Animals; Antagomirs; Blood-Brain Barrier; Brain; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Male; MicroRNAs; Neurons; Rats; Rats, Wistar; Sirolimus; Stroke

Reperfusion injury exists as the major obstacle to full recovery of neuron functions after ischemic stroke onset and clinical thrombolytic therapies. Complex cellular cascades including oxidative stress, neuroinflammation, and brain vascular impairment occur within neurovascular units, leading to microthrombus formation and ultimate neuron death. In this work, a multitarget micelle system is developed to simultaneously modulate various cell types involved in these events. Briefly, rapamycin is encapsulated in self-assembled micelles that are consisted of reactive oxygen species (ROS)-responsive and fibrin-binding polymers to achieve micelle retention and controlled drug release within the ischemic lesion. Neuron survival is reinforced by the combination of micelle facilitated ROS elimination and antistress signaling pathway interference under ischemia conditions. In vivo results demonstrate an overall remodeling of neurovascular unit through micelle polarized M2 microglia repair and blood-brain barrier preservation, leading to enhanced neuroprotection and blood perfusion. This strategy gives a proof of concept that neurovascular units can serve as an integrated target for ischemic stroke treatment with nanomedicines.

Topics: Apoptosis; Blood-Brain Barrier; Brain; Brain Ischemia; Cell Line; Cerebrovascular Circulation; Humans; Micelles; Microcirculation; Microglia; Neurons; Neuroprotective Agents; Oligopeptides; Polyethylene Glycols; Reactive Oxygen Species; Sirolimus; Stroke; Thrombosis

Alpha-lipoic Acid(ALA), an endogenous short-chain fatty acid, has been found inducing a protective effect against ischemia and reperfusion(I/R) injury. Recently, mTOR signaling pathway has been proved to involve in the mechanism of I/R injury. In our previous study, we determined that ALA could protect cerebral endothelial cells against I/R injury via mTOR signaling pathway. However, whether ALA can protect against brain I/R injury in vivo and its mechanisms is uncertain. In this study, we try to explore if the ALA treatment can protect against brain I/R injury and confirm the relationship between ALA and mTOR signaling pathway. ALA was administrated to the animals after dMCAo and reperfusion model established with or without rapamycin pre-treatment. The results showed the infarct size was obviously reduced after ALA treatment in acute stage, neurological functions were also improved distinctly. The mTOR signaling pathway was remarkably blocked after brain I/R injury while it could be activated through ALA treatment. However, rapamycin, can abolish the protective effects induced by ALA treatment in both acute and long-term phase. In conclusion, we demonstrate the protective effects induced by ALA treatment against the brain I/R injury in rats and mTOR signaling pathway is required for the protective effects of ALA against brain I/R injury. The results might contribute to the potential clinical application of ALA and provide a potential therapeutic target on ischemic stroke.

Topics: Animals; Brain Ischemia; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sirolimus; Thioctic Acid; TOR Serine-Threonine Kinases

Stroke is considered an underlying etiology of the development of seizures. Stroke leads to glucose and oxygen deficiency in neurons, resulting in brain dysfunction and injury. Mild hypothermia is a therapeutic strategy to inhibit stroke‑induced seizures, which may be associated with the regulation of energy metabolism of the brain. Mammalian target of rapamycin (mTOR) signaling and solute carrier family 2, facilitated glucose transporter member (GLUT)‑1 are critical for energy metabolism. Furthermore, mTOR overactivation and GLUT‑1 deficiency are associated with genetically acquired seizures. It has been hypothesized that mTOR and GLUT‑1 may additionally be involved in seizures elicited by stroke. The present study established global cerebral ischemia (GCI) models of rats. Convulsive seizure behaviors frequently occurred during the first and the second days following GCI, which were accompanied with seizure discharge reflected in the EEG monitor. Expression of phosphor (p)‑mTOR and GLUT‑1 were upregulated in the cerebral cortex and hippocampus, as evidenced by immunohistochemistry and western blot analyses. Mild hypothermia and/or rapamycin (mTOR inhibitor) treatments reduced the number of epileptic attacks, seizure severity scores and seizure discharges, thereby alleviating seizures induced by GCI. Mild hypothermia and/or rapamycin treatments reduced phosphorylation levels of mTOR and the downstream effecter p70S6 in neurons, and the amount of GLUT‑1 in the cytomembrane of neurons. The present study revealed that mTOR is involved in stroke‑induced seizures and the anti‑seizure effect of mild hypothermia. The role of GLUT‑1 in stroke‑elicited seizures appears to be different from the role in seizures induced by other reasons. Further studies are necessary in order to elucidate the exact function of GLUT-1 in stroke‑elicited seizures.

Topics: Animals; Biomarkers; Brain Ischemia; Disease Models, Animal; Electroencephalography; Glucose; Glucose Transporter Type 1; Hypothermia, Induced; Immunohistochemistry; Male; Neurons; Rats; Seizures; Severity of Illness Index; Signal Transduction; Sirolimus; Stroke; TOR Serine-Threonine Kinases

Ischemic postconditioning (IPostC) protects against stroke, but few have studied the pathophysiological mechanisms of its long-term protective effects. Here, we investigated whether the mTOR pathway is involved in the long-term protective effects of IPostC. Stroke was induced in rats by distal middle cerebral artery occlusion (dMCAo) combined with 30 min of bilateral common carotid artery (CCA) occlusion, and IPostC was induced after the CCA release. Injury size and behavioral tests were measured up to 3 weeks post stroke. We used rapamycin and mTOR shRNA lentiviral vectors to inhibit mTOR activities, while S6K1 viral vectors, a main downstream mTOR gene, were used to promote mTOR activities. We found that rapamycin administration abolished the long-term protective effects of IPostC. In addition, IPostC promoted the presynaptic growth associated protein 43 (GAP-43) and the postsynaptic protein 95 (PSD-95) levels at 1 week post-stroke, which were reduced by rapamycin. Furthermore, rapamycin reduced phosphorylated mTOR (p-mTOR) protein levels measured at 3 weeks after stroke. These results were confirmed by mTOR shRNA transfection. Moreover, we found that injection of S6K1 viral vectors promoted GAP-43 and PSD-95 protein levels. We conclude that mTOR may play a crucial, protective role in brain damage after stroke and contribute to the protective effects of IPostC.

Topics: Animals; Brain; Brain Ischemia; Ischemic Postconditioning; Male; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Stroke; TOR Serine-Threonine Kinases

We evaluated the neuroprotective role of rapamycin, a mammalian target of rapamycin (mTOR) kinase inhibitor, in cerebral ischaemia and locomotor function in a mouse model of subarachnoid haemorrhage (SAH). Pretreatment with rapamycin, an mTOR kinase inhibitor, resulted in better recovery from cerebral hypoxia early after SAH than control (P < .05), while the values of peak flow velocity in the middle cerebral artery did not change significantly (P > .05). Average distance travelled and the ratio of central-area distance/total travelled distance determined by open-field test after day 14 was significantly higher in mice pretreated with rapamycin than in control mice (P < .05). Inhibition of the mTOR pathway could be protective against post-SAH early brain injury, ameliorating brain tissue hypoxia and locomotor hypoactivity.

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Male; Mice; Neuroprotective Agents; Sirolimus; Subarachnoid Hemorrhage; TOR Serine-Threonine Kinases

Stroke, as a kind of acute cerebrovascular diseases, has greatly influenced the patients' quality of life and left a huge public health burden. Vitexin is a flavone C-glycoside (apigenin-8-C-?-D-glucopyranoside) present in several medicinal and other plants. This study aims to explore the role of vitexin in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic stroke. The results showed that the MCAO-induced brain infarction was obviously decreased by vitexin. And the abnormal protein levels of Caspase-3, Bcl-2-associated X protein (Bax), antigen identified by monoclonal antibody (Ki-67) and B cell lymphoma 2 (Bcl-2) in MCAO model rats were reversed by vitexin. Further research indicated that vitexin alleviated MCAO-induced oxidative injury by reducing the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA) and nitric Oxide (NO). In addition, vitexin attenuated the secretion of pro-inflammatory cytokine (interleukin (IL)-6 and tumor necrosis factor alpha (TNF-?)) and increased anti-inflammatory cytokine (IL-10) production to ameliorate MCAO-induced inflammation. What's more, vitexin repressed the MCAO-induced autophagy through mechanistic target of rapamycin (mTOR)/Ulk1 pathway. Specifically, the MCAO-induced decreased expression of mTOR, peroxisome proliferator-activated receptor ? (PPAR?) and p62 were inhibited by vitexin. At the same time, MCAO-induced increased expression of Ulk1, Beclin1 and rate of LC3?/LC3? also were repressed by vitexin. But the inhibition of vitexin on the MCAO-induced oxidative injury, apoptosis and inflammation were reversed by rapamycin. These results implied that vitexin suppressed the autophagy dysfunction to attenuate MCAO-induced cerebral ischemic stroke via mTOR/Ulk1 pathway.

Topics: Animals; Apigenin; Apoptosis; Autophagy; Autophagy-Related Protein-1 Homolog; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Inflammation; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Sirolimus; Stroke; TOR Serine-Threonine Kinases

Cerebral ischemia/reperfusion (I/R) injury is a common pathological process after cardiac arrest, shock and acute cerebral infarction recanalization, which causes serious injury in brain function. Pinocembrin (Pino), a natural flavonoid at the highest concentration in propolis, exhibited a variety of biological effects, including antitumor, antimicrobial and anti-inflammatory activities. However, the effects of Pino on brain injured after I/R and the mechanisms of its neuroprotective effects remain elusive.. In the present study, we used I/R model rats underwent transient cerebral ischemia inducing by four-vessel occlusion and reperfusion. Pino alone or in combination with autophagy inducer rapamycin (RAPA) was administered to I/R rats. The behavior and cognitive function were evaluated by open field test and Morris water maze test. HE staining was used to determine the survival of hippocampus CA1 pyramidal cells. Three key proteins of autophagy, LC3, Beclin1 and p62, were detected by Western blot.. Our results showed that Pino could significantly reduce the damage of hippocampus CA1 pyramidal neurons and alleviate the impairments of behavior and cognitive function in I/R rats. Pino also decreased the expression of LC3II and Beclin1 and increased the level of p62 in hippocampus CA1 of I/R rats. In addition, Pino also decreased RAPA-induced neuronal damage and excessive activation of autophagy in I/R rats.. Taken together, these results suggested that Pino could protect the brain injury induced by I/R and the potential mechanisms might attribute to inhibition of autophagy activity.

Topics: Animals; Autophagy; Beclin-1; Behavior, Animal; Brain Ischemia; CA1 Region, Hippocampal; Cognition; Disease Models, Animal; Flavanones; Male; Maze Learning; Microtubule-Associated Proteins; Motor Activity; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Reperfusion Injury; Sequestosome-1 Protein; Sirolimus; Time Factors

To investigate whether rapamycin treatment starting at 24 h after cerebral ischemia/reperfusion(I/R) has protective effect on brain injury in rats.. The rat I/R model was established by middle cerebral artery occlusion according to Longa's method. A total of 104 Sprague Dawley rats were randomly divided into sham group, model group, and rapamycin-treated groups (6 h or 24 h after modeling). Neurological function was assessed with neurological severity score (NSS). Triphenyl tetrazolium chloride (TTC) staining and Fluoro-Jade B (FJB) staining were used to examine the infarct volume and neuronal apoptosis, respectively. The expression of p-S6 protein in mTOR signaling pathway was detected by Western blot analysis.. Compared with sham group, NSS of the model group was significantly increased and TTC staining indicated obvious infarct area (all. Rapamycin treatment starting at 24 h after I/R exhibits protective effect on brain injury in rats.

Topics: Animals; Brain Ischemia; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Treatment Outcome

Autophagy is an intracellular bulk self-degrading process in which cytoplasmic contents of abnormal proteins and excess or damaged organelles are sequestered into autophagosomes, and degraded upon fusion with lysosomes. Although autophagy is generally considered to be pro-survival, it also functions in cell death processes. We recently reported on the hippocampal, higher vulnerability to cerebral ischemia in mice lacking the circadian clock protein PERIOD1 (PER1), a phenomenon we found to be linked to a PER1-dependent modulation of the expression patterns of apoptotic/autophagic markers.. To exclude the contribution of vascular or glial factors to the innate vulnerability of Per1 knockout-mice (Per1-/--mice) to cerebral ischemia in vivo, we compared the autophagic machinery between primary hippocampal cultures from wild-type (WT)- and Per1-/--mice, using the lipophilic macrolide antibiotic, Rapamycin to induce autophagy.. Development of autophagy in WT cells involved an increased LC3-II-to-LC3-I ratio (microtubule-associated protein 1 light chain 3) and an overall increase in the level of LC3-II. In addition, immunostaining of LC3 in WT cells revealed the typical transformation of LC3 localization from a diffused staining to a dot- and ring-like pattern. In contrast, Per1-/--hippocampal cells were resistant to Rapamycin induced alterations of autophagy hallmarks.. Our in vitro data suggests that basal activity of autophagy seems to be modulated by PER1, and confirms the in vivo data by showing that the autophagic machinery is depressed in Per1-/--hippocampal neurons.The implication of both autophagy and circadian dysfunction in the pathogenesis of cerebral ischemia suggests that a functional connection between the two processes may exist.

Topics: Animals; Autophagy; Brain Ischemia; Disease Models, Animal; Gene Expression Regulation; Hippocampus; Immunosuppressive Agents; Lysosomal-Associated Membrane Protein 2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Neurons; Period Circadian Proteins; Sirolimus; Time Factors

Diabetes causes functional and structural changes in blood-brain barrier (BBB). The mammalian target of rapamycin (mTOR) has been associated with glucose metabolism, diabetes, and altering BBB permeability. Since there is only a narrow therapeutic window (3h) for stroke victims, it is important to investigate BBB disruption in the early stage of cerebral ischemia. We compared the degree of BBB disruption in diabetic and in control rats at two hours of reperfusion after one hour of middle cerebral artery (MCA) occlusion with or without inhibition of mTOR. Two weeks after streptozotocin ip to induce diabetes, MCA occlusion was performed. In half of the rats, an mTOR inhibitor, rapamycin was given for 2days before MCA occlusion. After one hour of MCA occlusion and two hours of the reperfusion, the transfer coefficient (K

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Capillary Permeability; Diabetes Mellitus, Experimental; Immunosuppressive Agents; Male; Rats; Rats, Inbred F344; Sirolimus

Hyperglycemia is known to exacerbate neuronal death resulted from cerebral ischemia. The mechanisms are not fully understood. The mammalian target of rapamycin (mTOR) pathway regulates cell growth, division and apoptosis. Recent studies suggest that activation of mTOR may mediate ischemic brain damage. The objective of the present experiment is to explore whether mTOR mediates ischemic brain damage in acute hyperglycemic animals. Rats were subjected to 10 min of forebrain ischemia under euglycemic, hyperglycemic and rapamycin-treated hyperglycemic conditions. The rat brain samples were collected from the cortex and hippocampi after 3h and 16h of reperfusion. The results showed that hyperglycemia significantly increased neuronal death in the cortex and hippocampus and the exacerbation effect of hyperglycemia was associated with further activation of mTOR under control and/or ischemic conditions. Inhibition of mTOR with rapamycin ameliorated the damage and suppressed hyperglycemia-elevated p-MTOR, p-P70S6K and p-S6. In addition, hyperglycemia

Topics: Animals; Brain Ischemia; Hyperglycemia; Immunosuppressive Agents; Male; Random Allocation; Rats; Rats, Wistar; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To investigate the effect of mammalian target of rapamycin(mTOR) inhibitor-rapamycin on cognitive function after chronic cerebral ischemia in mice and its molecular mechanism.. The chronic cerebral ischemia model was induced by ligation of right common carotid artery (rUCCAO) in 6-week-old ICR mice. The expressions of mTOR, S6K, S6 and corresponding phosphorylated proteins were detected by Western blotting at different time interval (1 h, 3 h, 6 h, 24 h, 3 d, 7 d, 2 w, 4 w, 6 w) after rUCCAO to determine the changes of mTOR signaling pathway. Rapamycin was administrated i.p. at the dose of 3.0 mg/kg 24 h after rUCCAO. Fluoro Jade B staining was used to detect the apoptotic cells. The expressions of Beclin and LC3-Ⅱ were detected by Western blotting to determine the status of autophagy. Morris water maze test and Y maze test were performed to evaluate cognitive functions.. The mTOR signaling pathway was abnormally activated from 6 h to 6 w after rUCCAO in mouse cortex. The activation of mTOR signaling pathway induced by rUCCAO was reversed by administration of rapamycin, and the apoptotic cell number was significantly decreased (146.1±16.3 vs 84.5±9.6,. Inhibiting mTOR pathway by rapamycin reverses the rUCCAO-induced cognitive impairment partly through the suppression of apoptosis and autophagy.

Topics: Animals; Brain Ischemia; Cognition; Enzyme Inhibitors; Mice; Mice, Inbred ICR; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Activation of the mammalian target of rapamycin (mTOR) leads to cell growth and survival. We tested the hypothesis that inhibition of mTOR would increase infarct size and decrease microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1h and reperfusion for 2h with and without rapamycin (20mg/kg once daily for two days prior to ischemia). Regional cerebral blood flow was determined using a C(14)-iodoantipyrine autoradiographic technique. Regional small-vessel arterial and venous oxygen saturations were determined microspectrophotometrically. The control ischemic-reperfused cortex had a similar blood flow and O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex. Rapamycin significantly increased cerebral O2 consumption and further reduced O2 supply/consumption balance in the reperfused area. This was associated with an increased cortical infarct size (13.5±0.8% control vs. 21.5±0.9% rapamycin). We also found that ischemia-reperfusion increased AKT and S6K1 phosphorylation, while rapamycin decreased this phosphorylation in both the control and ischemic-reperfused cortex. This suggests that mTOR is important for not only cell survival, but also for the control of oxygen balance after cerebral ischemia-reperfusion.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antipyrine; Blood Gas Analysis; Blood Pressure; Brain Ischemia; Carbon Isotopes; Cerebrovascular Circulation; Disease Models, Animal; Hemodynamics; Immunosuppressive Agents; Male; Oncogene Protein v-akt; Oxygen Consumption; Rats; Rats, Inbred F344; Reperfusion; Signal Transduction; Sirolimus; Time Factors

The mammalian target of rapamycin (mTOR) pathway is essential in neuronal survival and repair in cerebral ischemia. Decreases in blood-brain barrier (BBB) disruption are associated with a decrease in neuronal damage in cerebral ischemia. This study was performed to investigate how pre-inhibition of the mTOR pathway with rapamycin would affect BBB disruption and the size of the infarcted cortical area in the early stage of focal cerebral ischemia-reperfusion using quantitative analysis of BBB disruption. Rats were treated with 20mg/kg of rapamycin i.p. once a day for 2days (Rapamycin Group) or vehicle (Control Group) before transient middle cerebral artery (MCA) occlusion. After one hour of MCA occlusion and two hours of reperfusion, the transfer coefficient (Ki) of (14)C-α-aminoisobutyric acid ((14)C-AIB) to measure the degree of BBB disruption and the size of the cortical infarct were determined. Ischemia-reperfusion increased the Ki in the Rapamycin treated (+15%) as well as in the untreated control group (+13%). However, rapamycin pretreatment moderately decreased Ki in the contralateral (-30%) as well as in the ischemic-reperfused (-29%) cortex when compared with the untreated control group. Rapamycin pretreatment substantially increased the percentage of cortical infarct compared with the control group (+56%). Our data suggest that activation of mTOR pathway is necessary for neuronal survival in the early stage of cerebral ischemia-perfusion and that the reason for the enlarged cortical infarct by rapamycin pretreatment may be related to its non-BBB effects on the mTOR pathway.

Topics: Animals; Blood-Brain Barrier; Brain Infarction; Brain Ischemia; Cerebral Cortex; Male; Permeability; Rats, Inbred F344; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The objectives of the present study are to investigate the activation of mTOR and ERK1/2 signaling after cerebral ischemia in diabetic rats and to examine the neuroprotective effects of rapamycin. Ten minutes transient global cerebral ischemia was induced in straptozotocin-induced diabetic hyperglycemic rats and non-diabetic, euglycemic rats. Brain samples were harvested after 16 h of reperfusion. Rapamycin or vehicle was injected 1 month prior to the induction of ischemia. The results showed that diabetes increased ischemic neuronal cell death and associated with elevations of p-P70S6K and Ras/ERK1/2 and suppression of p-AMPKα. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and suppressed phosphorylation of P70S6K and ERK1/2. It is concluded that diabetes activates mTOR and ERK1/2 signaling pathways in rats subjected to transient cerebral ischemia and inhibition of mTOR by rapamycin reduces ischemic brain damage and suppresses the mTOR and ERK1/2 signaling in diabetic settings.

Topics: AMP-Activated Protein Kinases; Animals; Blood Glucose; Blotting, Western; Brain Ischemia; Cerebral Cortex; Diabetes Mellitus, Experimental; DNA Fragmentation; In Situ Nick-End Labeling; Male; MAP Kinase Signaling System; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases

It has been reported that cerebral ischemia/reperfusion (I/R) injury can activate autophagy. However, the role of autophagy in cerebral I/R injury remains controversy. Two major proteins, mTOR and Beclin-1, govern the formation of autophagosomes to regulate autophagy activity. However, the cross-talking between Beclin-1 and mTOR in cerebral I/R injury remains elusive. In this study, global cerebral I/R injury animal model and focal cerebral I/R injury animal model were induced to test the variation of Beclin-1 level in vivo. To further confirm the variation of Beclin-1 level and investigate the cross-talking between Beclin-1 and mammalian target of rapamycin (mTOR) in I/R injury, we used cobalt chloride (CoCl2) to develop an I/R injury cell model in HT22 cell line. Our data showed that the levels of Beclin-1 and phosphorylated mammalian target of rapamycin (p-mTOR) were clearly induced by I/R injury in vitro. And the time course studies suggested that the Beclin-1 and mTOR may have coordinated regulation in ischemia stages but not in reperfusion stages. Moreover, inhibitor of mTOR could prevent Beclin-1 decreasing, but this prevention may play opposite roles in different stages of I/R injury. We conclude that this study represents a major advance in our understanding of the cross-talking of two key proteins, Beclin-1 and mTOR, in autophagy and the role of autophagy in cerebral I/R injury.

Topics: Analysis of Variance; Animals; Antimutagenic Agents; Apoptosis Regulatory Proteins; Beclin-1; Brain Ischemia; Cell Line; Cell Survival; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Male; Mice; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

The aims of this study are to clarify the role of mTOR in mediating cerebral ischemic brain damage and the effects of rapamycin on ischemic outcomes. Ten minutes of forebrain ischemia was induced in rats, and their brains were sampled after 3 h, 16 h, and 7 days reperfusion for histology, immunohistochemistry and biochemical analysis. Our data demonstrated that cerebral ischemia resulted in both apoptotic and necrotic neuronal death; cerebral ischemia and reperfusion led to significant increases of mRNA and protein levels of p-mTOR and its downstream p-P70S6K and p-S6; elevation of LC3-II, and release of cytochrome c into the cytoplasm in both the cortex and hippocampus. Inhibition of mTOR by rapamycin markedly reduced ischemia-induced damage; suppressed p-Akt, p-mTOR, p-P70S6K and p-S6 protein levels; decreased LC3-II and Beclin-1; and prevented cytochrome c release in the two structures. All together, these data provide evidence that cerebral ischemia activates mTOR and autophagy pathways. Inhibition of mTOR deactivates the mTOR pathway, suppresses autophagy, prevents cytochrome c release and reduces ischemic brain damage.

Topics: Animals; Brain Ischemia; Cytochromes c; In Situ Nick-End Labeling; Prosencephalon; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin has been demonstrated to exhibit neuroprotective functions via the activation of autophagy in a cerebral ischemia model. However, the involvement of mitophagy in this process and its contribution to the protection of mitochondrial function remains unknown. The present study explored the characteristics of mitophagy after cerebral ischemia and the effect of rapamycin on mitochondrial function. Male Sprague-Dawley rats underwent transient middle cerebral artery occlusion (tMCAO). Neurological deficits scores; infarct volumes; mitophagy morphology; and the levels of malondialdehyde (MDA), adenosine triphosphate (ATP) and mitochondrial membrane potentials (Δψm) were examined. The expression of LC3, Beclin-1 and p62 in the mitochondrial fraction combined with transmission electronic microscopy were used to explore mitophagic activity after ischemia. We also blocked autophagosome formation using 3-methyladenine (3-MA) to check the linkage between the mitochondrial protective effect of rapamycin and enhanced mitophagy. We observed that rapamycin significantly enhanced mitophagy, as evidenced by the increase in LC3-II and Beclin-1 expression in the mitochondria and p62 translocation to the mitochondria. Rapamycin reduced infarct volume, improved neurological outcomes and inhibited mitochondrial dysfunction compared with the control animals (p<0.05). However, these protective effects were reversed by 3-methyladenine treatment after rapamycin. The present study indicates that rapamycin treatment attenuates mitochondrial dysfunction following cerebral ischemia, which is linked to enhanced mitophagy.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphate; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Blotting, Western; Brain; Brain Ischemia; Immunohistochemistry; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Male; Malondialdehyde; Membrane Potential, Mitochondrial; Membrane Proteins; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Mitochondria; Mitophagy; Protein Transport; Rats; Rats, Sprague-Dawley; Sequestosome-1 Protein; Sirolimus; Stroke

To explore the effects of modulating autophagy on neuroamyloidogenesis in an ischemic stroke model of cultured neuroblastoma 2a (N2a)/Amyloid precursor protein (APP)695 cells.. The ischemic stroke model of N2a/APP695 cells was made by 6h oxygen-glucose deprivation/12h reperfusion (OGDR). Drug administration of 3-methyladenine (3-MA), rapamycin or dl-3-n-butylphthalide (NBP) was started at the beginning of the OGDR and lasted until the end of reperfusion, in order to explore their effects on N2a/APP695 cells under OGDR conditions. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 12h. Cell viability and injury were investigated. The key proteins of nuclear factor kappa B (NF-κB) pathway and a key component of autophagy Beclin 1 were detected by Western blotting; immunofluorescence double-staining of amyloid-β (Aβ)1-42 with Beclin 1 was performed to investigate their cellular co-localization relationship; β-secretase and γ-secretase activity assay and Aβ1-42 enzyme-linked immunosorbent assay were performed to investigate the amyloidogenesis.. The results showed that, OGDR enhanced cell injury, autophagy activity, neuroinflammation and Aβ generation in N2a/APP695 cells; down-regulating autophagy by 3-MA and NBP increased cell viability, decreased lactate dehydrogenase (LDH) production, inhibited the activation of NF-κB pathway, suppressed β- and γ-secretase activities and Aβ generation; while up-regulating autophagy by rapamycin got the opposite results; immunofluorescence double-staining results showed elevated Aβ1-42(+) signal was co-localized with Beclin 1(+) signal.. Our data suggested that down-regulating autophagy may inhibit ischemia-induced neuroamyloidogenesis via suppressing the activation of NF-κB pathway. This study might help us to find a new therapeutic strategy to prevent brain ischemic damage and depress the risk of post-stroke dementia.

Topics: Adenine; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Autophagy; Benzofurans; Brain Ischemia; Cell Survival; Mice; Neuroblastoma; Sirolimus; Stroke; Tumor Cells, Cultured

Brain edema is a major consequence of cerebral ischemia reperfusion. However, few effective therapeutic options are available for retarding the brain edema progression after cerebral ischemia. Recently, rapamycin has been shown to produce neuroprotective effects in rats after cerebral ischemia reperfusion. Whether rapamycin could alleviate this brain edema injury is still unclear. In this study, the rat stroke model was induced by a 1-h left transient middle cerebral artery occlusion using an intraluminal filament, followed by 48 h of reperfusion. The effects of rapamycin (250 μg/kg body weight, intraperitoneal; i.p.) on brain edema progression were evaluated. The results showed that rapamycin treatment significantly reduced the infarct volume, the water content of the brain tissue and the Evans blue extravasation through the blood-brain barrier (BBB). Rapamycin treatment could improve histological appearance of the brain tissue, increased the capillary lumen space and maintain the integrity of BBB. Rapamycin also inhibited matrix metalloproteinase 9 (MMP9) and aquaporin 4 (AQP4) expression. These data imply that rapamycin could improve brain edema progression after reperfusion injury through maintaining BBB integrity and inhibiting MMP9 and AQP4 expression. The data of this study provide a new possible approach for improving brain edema after cerebral ischemia reperfusion by administration of rapamycin.

Topics: Animals; Aquaporin 4; Blood-Brain Barrier; Brain; Brain Edema; Brain Ischemia; Cerebrovascular Circulation; Male; Matrix Metalloproteinase 9; Permeability; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Signaling by the mammalian target of rapamycin (mTOR) plays an important role in the modulation of both innate and adaptive immune responses. However, the role and underlying mechanism of mTOR signaling in poststroke neuroinflammation are largely unexplored. In this study, we injected rapamycin, a mTOR inhibitor, by the intracerebroventricular route 6 h after focal ischemic stroke in rats. We found that rapamycin significantly reduced lesion volume and improved behavioral deficits. Notably, infiltration of γδ T cells and granulocytes, which are detrimental to the ischemic brain, was profoundly reduced after rapamycin treatment, as was the production of proinflammatory cytokines and chemokines by macrophages and microglia. Rapamycin treatment prevented brain macrophage polarization toward the M1 type. In addition, we also found that rapamycin significantly enhanced anti-inflammation activity of regulatory T cells (Tregs), which decreased production of proinflammatory cytokines and chemokines by macrophages and microglia. Depletion of Tregs partially elevated macrophage/microglia-induced neuroinflammation after stroke. Our data suggest that rapamycin can attenuate secondary injury and motor deficits after focal ischemia by enhancing the anti-inflammation activity of Tregs to restrain poststroke neuroinflammation.

Topics: Animals; Brain Ischemia; Immunosuppressive Agents; Inflammation; Macrophages; Male; Microglia; Rats; Rats, Sprague-Dawley; Receptors, Antigen, T-Cell, gamma-delta; Signal Transduction; Sirolimus; Stroke; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

Remote ischemic perconditioning (RIPer) has been proved to provide potent cardioprotection. However, there are few studies on neuroprotection of RIPer. This study aims to clarify the neuroprotective effect of RIPer and the role of autophagy induced by RIPer against cerebral ischemia reperfusion injury in rats. Using a transient middle cerebral artery occlusion (MCAO) model in rats to imitate focal cerebral ischemia. RIPer was carried out 4 cycles of 10 min ischemia and 10 min reperfusion, with a thin elastic band tourniquet encircled on the bilateral femoral arteries at the start of 10 min after MCAO. Autophagy inhibitor 3-methyladenine (3-MA) and autophagy inducer rapamycin were administered respectively to determine the contribution of autophagy in RIPer. Neurologic deficit scores, infarct volume, brain edema, Nissl staining, TUNEL assay, immunohistochemistry and western blot was performed to analyze the neuroprotection of RIPer and the contribution of autophagy in RIPer. RIPer significantly exerted neuroprotective effects against cerebral ischemia reperfusion injury in rats, and the autophagy-lysosome pathway was activated by RIPer treatment. 3-MA reversed the neuroprotective effects induced by RIPer, whereas rapamycin ameliorated the brain ischemic injury. Autophagy activation contributes to the neuroprotection by RIPer against focal cerebral ischemia in rats.

Topics: Adenine; Animals; Autophagy; Brain Ischemia; Cerebral Infarction; Ischemic Postconditioning; Ischemic Preconditioning; Male; Models, Animal; Neuroprotective Agents; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

The immunosuppressant drug rapamycin was reported to have an antiaging activity, which was attributed to the TORC1 inhibition that inhibits cell proliferation and increases autophagy. However, rapamycin also exhibits a number of harmful adverse effects. Whether rapamycin can be developed into an antiaging agent remains unclear.. We demonstrated that rapamycin at micro-doses (below the TORC1 inhibiting concentration) exhibits a cell-protective activity: (1) It protects cultured neurons against neurotoxin MPP(+) and H2O2. (2) It increases survival time of neuron in culture. (3) It maintains the nonproliferative state of cultured senescent human fibroblasts and prevents cell death induced by telomere dysfunction. (4) In animal models, it decreased the cerebral infarct sizes induced by acute ischemia and dramatically extended the life span of stroke prone spontaneously hypertensive rats (SHR-SPs).. We propose that rapamycin at micro-dose can be developed into an antiaging agent with a novel mechanism.

Topics: Aging; Animals; beta-Galactosidase; Brain Infarction; Brain Ischemia; Cell Death; Cell Line, Transformed; Cerebral Cortex; Disease Models, Animal; Fibroblasts; Humans; Hydrogen Peroxide; Immunosuppressive Agents; Male; Mice, Inbred C57BL; Phosphorylation; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Tacrolimus

Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain Ischemia; Caspase 3; Cerebrum; Immunosuppressive Agents; In Situ Nick-End Labeling; Ischemic Preconditioning; Male; Nerve Degeneration; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5-20 mM) and bafilomycin A 1 (75-150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50-200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain Ischemia; Caspase 12; Caspase 3; Cells, Cultured; Cerebral Cortex; Cinnamates; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Mice; Neurons; Oxygen; Rats; Rats, Sprague-Dawley; Sirolimus; Thiourea; Transcription Factor CHOP

Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.. A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.. The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain; Brain Ischemia; Immunosuppressive Agents; Ischemic Postconditioning; Male; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Sirolimus; Up-Regulation

Stroke is a major cause of mortality and disability. The management with thrombolytic therapy has to be initiated within 3-4 h and is associated with limitations like increased risk of intracranial hemorrhage and progression of cerebral injury. Immunophilin inhibitors such as cyclosporine A and tacrolimus have been shown to afford neuroprotection by improving neurological functions and infarct volume in models of ischemic stroke. In the present study, the effect of rapamycin in middle cerebral artery occlusion (MCAo) model of ischemic stroke was evaluated. Ischemic stroke was induced in rats by occluding the MCA using the intraluminal thread. After 1 h of MCAo, animals were administered rapamycin (50, 150, 250 μg/kg, i.p.). After 2 h of occlusion, reperfusion was done. Thirty minutes after reperfusion, animals were subjected to diffusion-weighted magnetic resonance imaging for assessment of protective effect of rapamycin. Twenty-four hours after MCAo, motor performance was assessed, the animals were euthanized and the brains were removed for estimation of malondialdehyde, glutathione, nitric oxide and myeloperoxidase. Significant improvement was observed with rapamycin 150 and 250 μg/kg in percent infarct area, apparent diffusion coefficient and signal intensity as compared to vehicle treated group. Rapamycin treatment ameliorated motor impairment associated with MCAo and significantly reversed the changes in levels of malondialdehyde, glutathione, nitric oxide and myeloperoxidase. The results of the present study indicate neuroprotective effect of rapamycin in MCAo model of stroke. Therefore, rapamycin might be considered as a therapeutic strategy for stroke management.

Topics: Animals; Brain; Brain Ischemia; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Dose-Response Relationship, Drug; Glutathione; Hand Strength; Humans; Infarction, Middle Cerebral Artery; Male; Malondialdehyde; Neuroimaging; Neuroprotective Agents; Nitric Oxide; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Rotarod Performance Test; Sirolimus

We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI) rapamycin administration increases autophagy, decreases apoptosis and significantly reduces brain damage. After HI, when autophagy is blocked neuronal cells rapidly progress toward necrotic cell death. The present study was undertaken to assess the potential role of activation of autophagic and phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathways in the neuroprotective effect of rapamycin. Rapamycin administration caused a significant reduction of 70 kDa S6 kinase (p70S6K) phosphorylation and a significant increase of the autophagic proteins Beclin 1 and microtubule-associated protein 1 light chain 3 (LC3), as of monodansylcadaverine (MDC) labeling in the lesioned side. The phosphorylation of Akt and cAMP response element binding protein (CREB) was increased in neuronal cells, and both p-Akt and p-CREB colocalized with Beclin 1. Wortmannin (WM) administration significantly reduced Akt and CREB phosphorylation as well as the neuroprotective effect of rapamycin but did not affect the phosphorylation of p70S6K, the expression of Beclin 1 and LC3, and MDC labeling. In contrast, 3-methyladenine (3MA) reduced the increased Beclin 1 expression, the MDC labeling and the neuroprotective effect of rapamycin without affecting Akt phosphorylation. However, both compounds significantly increased necrotic cell death. Taken together, these data indicate that in neonatal HI autophagy can be part of an integrated prosurvival signaling which includes the PI3K-Akt-mammalian target of rapamycin (mTOR) axis. When the autophagic or the PI3K-Akt-mTOR pathways are interrupted cells undergo necrotic cell death.

Topics: Androstadienes; Animals; Animals, Newborn; Antibiotics, Antineoplastic; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain Ischemia; Cyclic AMP Response Element-Binding Protein; Humans; Hypoxia; Microtubule-Associated Proteins; Neurons; Neuroprotective Agents; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Wortmannin

Several recent studies have showed that autophagy is involved in ischemic brain damage, but it may also play a pro-survival role in ischemic preconditioning. This study was taken to determine the role of autophagy in an animal model of cerebral ischemic preconditioning (IPC). Focal cerebral IPC was produced in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (Baf A1) or the autophagy inducer rapamycin to evaluate the contribution of autophagy to IPC-induced neuroprotection. The results from electron microscopic examinations and immunofluorescence showed that both IPC and PFI induced autophagy activation, but the extent and persistence of autophagy activation were varied. IPC treatment significantly reduced infarct volume, brain edema and motor deficits after subsequent PFI, whereas 3-MA and Baf A1 suppressed the neuroprotection induced by IPC. 3-MA pretreatment also significantly attenuated upregulation of LC3-II, beclin 1 and HSP70 and downregulation of p62. To further determine if autophagy induction is responsible for IPC-induced neuroprotection, rats were treated with rapamycin 24 h before the onset of PFI. The results showed that rapamycin reduced infarct volume, brain edema and motor deficits induced by PFI. Rapamycin pretreatment also increased the protein levels of LC3-II and beclin 1. These results demonstrate that autophagy activation during IPC offers a remarkable tolerance to a subsequent fatal ischemic insult, and IPC's neuroprotective effects can be mimicked by autophagy inducers.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain Ischemia; Disease Models, Animal; Down-Regulation; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Microtubule-Associated Proteins; Neostriatum; Neurogenesis; Neurons; Neuroprotective Agents; Phagosomes; Rats; Rats, Sprague-Dawley; Sequestosome-1 Protein; Sirolimus; Up-Regulation

The two complexes of the mammalian target of rapamycin (mTOR), mTORC1 and mTORC2, have central functions in the integration of both extracellular and intracellular signals that are also critical players in the induction of post-ischemic long-term potentiation (i-LTP), a pathological form of plasticity inducible in striatal medium spiny neurons (MSNs) after a brief episode of in vitro ischemia. To evaluate the involvement of mTOR complexes during ischemia we analyzed the time course of i-LTP by intracellular recordings of MSNs from corticostriatal slices incubated with 1μM mTOR inhibitor rapamycin. Although rapamycin did not affect the amplitude and duration of ischemia-induced membrane depolarization it fully prevented i-LTP, leaving unaffected the capability to undergo activity-dependent LTP following high-frequency stimulation of corticostriatal fibers. The present results argue for a role of mTOR complex in i-LTP and suggest that rapamycin, by selectively blocking i-LTP, represents a promising therapeutic tool to limit cellular damage after ischemic brain insult.

Topics: Animals; Biophysics; Brain Ischemia; Corpus Striatum; Disease Models, Animal; Electric Stimulation; Excitatory Postsynaptic Potentials; Glucose; Hypoxia; Intracellular Signaling Peptides and Proteins; Long-Term Potentiation; Male; Neurons; Patch-Clamp Techniques; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.

Topics: Animals; Brain Ischemia; Cell Cycle; Ciclopirox; Dose-Response Relationship, Drug; Flavonoids; Growth Inhibitors; Male; Nerve Growth Factor; Neurites; PC12 Cells; Piperidines; Pyridones; Pyrrolidines; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus

The immunosuppressive action of the drug FK506 involves inhibition of calcineurin in T-lymphocytes by a complex of FK506 and an FK506 binding protein, FKBP12, a member of the immunophilin protein family. The functional role of brain immunophilins is, however, unclear. We show here that FK506 is a powerful neuroprotective agent in an in vivo model of focal cerebral ischaemia when administered up to 60 min post-occlusion. The minimum effective neuroprotective dose is comparable with the immunosuppressant dose in humans, suggesting that FK506 may have clinical potential for the treatment of stroke. Although the related immunosuppressants rapamycin and cyclosporin failed to reduce brain damage, the finding that rapamycin pretreatment blocked the effect of FK506 confirms a role for immunophilins in the neuroprotective mechanism.

Topics: Animals; Brain Ischemia; Carrier Proteins; Cerebral Cortex; Corpus Striatum; Cyclosporine; Heat-Shock Proteins; Humans; Immunosuppressive Agents; Male; Polyenes; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins