Neuroprotection by the soy isoflavone, genistein, via inhibition of mitochondria-dependent apoptosis pathways and reactive oxygen induced-NF-κB activation in a cerebral ischemia mouse model

Recently, the treatment of stroke has focused on antioxidant therapies, where oxidative stress is implicated. The preventive and therapeutic potential of plant compounds on ischemic stroke has been intensively studied because many of them contain antioxidant properties. Genistein, one of the active ingredients in soybean, possesses many bioactivities. In this study, we investigated the potential neuroprotective effects of genistein and its possible mechanism of action in a cerebral ischemia mouse model. Mice were pretreated with genistein (2.5, 5, and 10mg/kg) or vehicle orally once daily for 14 consecutive days before transient middle cerebral artery occlusion was performed. Genistein at doses of 2.5-10mg/kg significantly reduced the infarct volume, improved the neurological deficit and prevented cell apoptosis after ischemia. In addition, genistein pretreatment was shown to inhibit the ischemia-induced reactive oxygen species (ROS) production, enhance the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), and decrease levels of malondialdehyde (MDA) in stroke mice. Moreover, genistein reversed the mitochondria dysfunction after ischemia, as evidenced by decreasing mitochondria ROS levels, preventing cytochrome C release to the cytoplasm and inhibiting caspase-3 activation. Western blotting showed ischemia activated the ROS-dependent nuclear factor-κB (NF-κB) signaling pathway, and genistein suppressed phosphorylation and activation of the NF-κB p65 subunit, as well as the phosphorylation and degradation of the inhibitor protein of κBα (IκBα). Our findings suggested that genistein has a neuroprotective effect in transient focal ischemia, which may involve regulation of mitochondria-dependent apoptosis pathways and suppression of ROS-induced NF-κB activation.     

Neuroprotective effect of glutamate-substituted analog of gramicidin A is mediated by the uncoupling of mitochondria

BackgroundReactive oxygen species are grossly produced in the brain after cerebral ischemia and reperfusion causing neuronal cell death. Mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Therefore, limited uncoupling of oxidative phosphorylation could be beneficial for cells exposed to deleterious oxidative stress-associated conditions by preventing excessive generation of reactive oxygen species.MethodsProtonophoric and uncoupling activities of different peptides were measured using pyranine-loaded liposomes and isolated mitochondria. To evaluate the effect of glutamate-substituted analog of gramicidin A ([Glu1]gA) administration on the brain ischemic damage, we employed the in vitro model of neuronal hypoxia using primary neuronal cell cultures and the in vivo model of cerebral ischemia induced in rats by the middle cerebral artery occlusion.Results[Glu1]gA was the most effective in proton-transferring activity among several N-terminally substituted analogs of gramicidin A tested in liposomes and rat brain and liver mitochondria. The peptides were found to be protective against ischemia-induced neuronal cell death and they lowered mitochondrial membrane potential in cultured neurons and diminished reactive oxygen species production in isolated brain mitochondria. The intranasal administration of [Glu1]gA remarkably diminished the infarct size indicated in MR-images of a brain at day 1 after the middle cerebral artery occlusion. In [Glu1]gA-treated rats, the ischemia-induced brain swelling and behavioral dysfunction were significantly suppressed.ConclusionsThe glutamate-substituted analogs of gramicidin A displaying protonophoric and uncoupling activities protect neural cells and the brain from the injury caused by ischemia/reperfusion.General significance[Glu1]gA may be potentially used as a therapeutic agent to prevent neuron damage after stroke.     

Neuroprotective effects of VEGF administration after focal cerebral ischemia/reperfusion: dose response and time window

Administration of vascular endothelial growth factor (VEGF) has been shown to increase cerebral blood flow and reduce neurological damage after experimental ischemic brain injury. The purpose of this study was to examine the optimal dose and time window for the neuroprotective effect of VEGF when administrated after focal ischemia/reperfusion injury in rabbits. Focal cerebral ischemia/reperfusion was induced by the middle cerebral artery occlusion (MCAO) method. In a dose response experiment, low (1.25 ng/μL), middle (2.5 ng/μL) and high (5.0 ng/μL) doses of VEGF were administered 2h after MCAO by the route of perifocal region. The VEGF at a dose of middle (2.5 ng/μL) displayed excellent effects on neuroprotective efficacy for focal cerebral ischemia/reperfusion injury. In another experiment, 2.5 ng/μL VEGF was administered at times varying from 2 to 8h after MCAO. Infarct volume, water content and neurological deficits were significantly reduced when VEGF was given at 2 and 3h after injury. The protective effect was less when the same dose was given at the later times. Thus, the present findings indicated that VEGF reduced ischemic neuronal danger with a therapeutic time window within the first 3h of transient MCAO and may be useful in the treatment of acute ischemic stroke in humans.     

Delayed injection of neural progenitor cells improved spatial learning dysfunction after cerebral ischemia

Although stem cells are likely to improve neurological deficits seen after cerebral ischemia, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning dysfunction remain to be clarified. We tested whether the delayed injection of exogenous NPCs could prevent learning dysfunction after cerebral ischemia. Cerebral ischemia was produced by the injection of microspheres into the right hemisphere of each rat. Injection of NPCs obtained from green fluorescent protein transgenic rats into the hippocampus on Day 7 after the induction of cerebral ischemia improved the modified neurological severity score and reduced the prolongation of the escape latency seen in the water maze task. A few of the injected NPCs were positive for mature neuronal markers. In addition, the injected NPCs expressed BDNF on Day 28 after cerebral ischemia. Thus, the exogenous NPCs delivered by injection could act as a source of neurotrophic factors and prevent cerebral ischemia-induced learning dysfunction.     

Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat

Stroke is a life-threatening disease with major cause of mortality and morbidity worldwide. The neuronal damage following cerebral ischemia is a serious risk to stroke patients. Oxidative stress and apoptotic damage play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. The objective of this study was to test the hypothesis that administration of edaravone (Edv) maintains antioxidant status in brain, improves the cholinergic dysfunction and suppresses the progression of apoptosis response in rat. To test this hypothesis, male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) of 2 h followed by reperfusion for 22 h. Edv was administered (10 mg/kg bwt) intraperitoneally 30 min before the onset of ischemia and 1 h after reperfusion. After reperfusion, rats were tested for neurobehavioral activities and were sacrificed for the infarct volume, estimation of oxidative damage markers. Edv treatment significantly reduced ischemic lesion volume, improved neurological deficits, contended oxidative loads, and suppressed apoptotic damage. In conclusion, treatment with Edv ameliorated the neurological and histological outcomes with elevated endogenous anti-oxidants status as well as reduced induction of apoptotic responses in MCA occluded rat. We theorized that Edv is among the pharmacological agents that reduce free radicals and its associated cholinergic dysfunction and apoptotic damage and have been found to limit the extent of brain damage following stroke.     

Estradiol rescues neurons from global ischemia-induced cell death: Multiple cellular pathways of neuroprotection

The potential neuroprotective role of sex hormones in chronic neurodegenerative disorders and acute brain ischemia following cardiac arrest and stroke is of a great therapeutic interest. Long-term pretreatment with estradiol and other estrogens affords robust neuroprotection in male and female rodents subjected to focal and global ischemia. However, the receptors (e.g., cell surface or nuclear), intracellular signaling pathways and networks of estrogen-regulated genes that intervene in neuronal apoptosis are as yet unclear. We have shown that estradiol administered at physiological levels for two weeks before ischemia rescues neurons destined to die in the hippocampal CA1 and ameliorates ischemia-induced cognitive deficits in ovariectomized female rats. This regimen of estradiol treatment involves classical intracellular estrogen receptors, transactivation of IGF-1 receptors and stimulation of the ERK/MAPK signaling pathway, which in turn maintains CREB activity in the ischemic CA1. We also find that a single, acute injection of estradiol administrated into the brain ventricle immediately after an ischemic event reduces both neuronal death and cognitive deficits. Because these findings suggest that hormones could be used to treat patients when given after brain ischemia, it is critical to determine whether the same or different pathways mediate this form of neuroprotection. We find that an agonist of the membrane estrogen receptor GPR30 mimics short latency estradiol facilitation of synaptic transmission in the hippocampus. Therefore, we are testing the hypothesis that GPR30 may act together with intracellular estrogen receptors to activate cell signaling pathways to promote neuron survival after global ischemia.     

Early Treatment with Poly(ADP-Ribose) Polymerase-1 Inhibitor (JPI-289) Reduces Infarct Volume and Improves Long-Term Behavior in an Animal Model of Ischemic Stroke

In patients with stroke and neurodegenerative diseases, overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) causes harmful effects by inducing apoptosis, necrosis, neuroinflammation, and immune dysregulation. The current study investigated the neuroprotective effect of a novel PARP-1 inhibitor, JPI-289, in an animal model of ischemic stroke. A transient middle cerebral artery occlusion (tMCAO, 2 h) model was used to determine the therapeutic effect and the most effective dose and time window of administration of JPI-289. We also investigated the long-term outcomes of treatment with JPI-289 by diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) MRI and by measuring neurological function at 24 h, 7 days, and 28 days after MCAO. The most effective dose and time window of administration of JPI-289 was 10 mg/kg administered 2 h after MCAO with reperfusion. Twenty-four hours after MCAO, infarct volume was reduced by 53% and the number of apoptotic cells was reduced by 56% compared with control. JPI-289 also reduced infarct volume by 16% in the permanent MCAO model. In an MRI-based study, initial infarct volume, as measured using DWI, was similar in the control and JPI-289-treated groups. However, infarct volume and brain swelling were significantly reduced in the group treated with JPI-289 (2 h) at 24 h and 7 days after MCAO. Neurological functions also improved in the group treated with JPI-289 (2 h) until 28 days after MCAO. Inhibition of PARP-1 has neuroprotective effects (reduction of infarct volume and brain swelling) in both tMCAO and pMCAO models of ischemic stroke.     

SM-31900, a novel NMDA receptor glycine-binding site antagonist,reduces infarct volume induced by permanent middle cerebral artery occlusion in spontaneously hypertensive rats

The purpose of this study was to investigate the effect of (3S)-7-chloro-3-[2-((1R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900), an antagonist with high selectivity and affinity for the NMDA receptor glycine-binding site, on the cerebral infarct volume in a permanent middle cerebral artery occlusion (MCAo) model, which was constructed by electrocoagulation of a unilateral middle cerebral artery distal to the olfactory tract using spontaneously hypertensive rats (SHRs). To investigate the dose-response characteristics and the therapeutic time window of SM-31900 in this MCAo model, we conducted three experiments, in which the administration of SM-31900 was started 5min (experiment I), 30min (experiment II), or 60min (experiment III) after MCAo, respectively. In all the studies, SM-31900 was administered by intravenous bolus injection followed by continuous intravenous infusion to obtain a steady-state level of this compound in blood immediately after its administration. The treatment with SM-31900 was continued until 24h after MCAo, at which time the cerebral infarct volume was measured. In experiment I, SM-31900 significantly reduced the infarct volume by 37% at a dosage of 0.38mg/kg bolus followed by 1.5mg/kg/h continuous infusion (0.38mg/kg+1.5mg/kg/h). In experiment II, the neuroprotective effect of SM-31900 was also significant, with a 25% reduction in infarct volume at a dosage of 0.38mg/kg+1.5mg/kg/h, and a 40% reduction at 1.5mg/kg+6.0mg/kg/h. Furthermore, even in experiment III, SM-31900 exerted a significant neuroprotective effect, with a 20% reduction at 1.5mg/kg+6.0mg/kg/h. These studies revealed that SM-31900 can exert a neuroprotective effect when it is administered up to at least 60min after the onset of ischemia in the MCAo model, an animal model of stroke, indicating that SM-31900 is a good candidate for treating acute brain ischemia.     

Intranasal guanosine administration presents a wide therapeutic time window to reduce brain damage induced by permanent ischemia in rats

In addition to its intracellular roles, the nucleoside guanosine (GUO) also has extracellular effects that identify it as a putative neuromodulator signaling molecule in the central nervous system. Indeed, GUO can modulate glutamatergic neurotransmission, and it can promote neuroprotective effects in animal models involving glutamate neurotoxicity, which is the case in brain ischemia. In the present study, we aimed to investigate a new in vivo GUO administration route (intranasal, IN) to determine putative improvement of GUO neuroprotective effects against an experimental model of permanent focal cerebral ischemia. Initially, we demonstrated that IN [³H] GUO administration reached the brain in a dose-dependent and saturable pattern in as few as 5 min, presenting a higher cerebrospinal GUO level compared with systemic administration. IN GUO treatment started immediately or even 3 h after ischemia onset prevented behavior impairment. The behavior recovery was not correlated to decreased brain infarct volume, but it was correlated to reduced mitochondrial dysfunction in the penumbra area. Therefore, we showed that the IN route is an efficient way to promptly deliver GUO to the CNS and that IN GUO treatment prevented behavioral and brain impairment caused by ischemia in a therapeutically wide time window.     

MMP-9 Inhibition: a Therapeutic Strategy in Ischemic Stroke

Ischemic stroke is a leading cause of disability worldwide. In cerebral ischemia there is an enhanced expression of matrix metallo-proteinase-9 (MMP-9), which has been associated with various complications including excitotoxicity, neuronal damage, apoptosis, blood–brain barrier (BBB) opening leading to cerebral edema, and hemorrhagic transformation. Moreover, the tissue plasminogen activator (tPA), which is the only US-FDA approved treatment of ischemic stroke, has a brief 3 to 4 h time window and it has been proposed that detrimental effects of tPA beyond the 3 h since the onset of stroke are derived from its ability to activate MMP-9 that in turn contributes to the breakdown of BBB. Therefore, the available literature suggests that MMP-9 inhibition can be of therapeutic importance in ischemic stroke. Hence, combination therapies of MMP-9 inhibitor along with tPA can be beneficial in ischemic stroke. In this review we will discuss the current status of various strategies which have shown neuroprotection and extension of thrombolytic window by directly or indirectly inhibiting MMP-9 activity. In the introductory part of the review, we briefly provide an overview on ischemic stroke, commonly used models of ischemic stroke and a role of MMP-9 in ischemia. In next part, the literature is organized as various approaches which have proven neuroprotective effects through direct or indirect decrease in MMP-9 activity, namely, using biotherapeutics, involving MMP-9 gene inhibition using viral vectors; using endogenous inhibitor of MMP-9, repurposing of old drugs such as minocycline, new chemical entities like DP-b99, and finally other approaches like therapeutic hypothermia.     

Delayed Treatment with Systemic (S)-Roscovitine Provides Neuroprotection and Inhibits In Vivo CDK5 Activity Increase in Animal Stroke Models

Background

Although quite challenging, neuroprotective therapies in ischemic stroke remain an interesting strategy to counter mechanisms of ischemic injury and reduce brain tissue damage. Among potential neuroprotective drug, cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates. Increasing evidence indisputably links cell cycle CDKs and CDK5 to the pathogenesis of stroke. Although recent studies have demonstrated promising neuroprotective efficacies of pharmacological CDK inhibitors in related animal models, none of them were however clinically relevant to human treatment.

Methodology/Principal Findings

In the present study, we report that systemic delivery of (S)-roscovitine, a well known inhibitor of mitotic CDKs and CDK5, was neuroprotective in a dose-dependent manner in two models of focal ischemia, as recommended by STAIR guidelines. We show that (S)-roscovitine was able to cross the blood brain barrier. (S)-roscovitine significant in vivo positive effect remained when the compound was systemically administered 2 hrs after the insult. Moreover, we validate one of (S)-roscovitine in vivo target after ischemia. Cerebral increase of CDK5/p25 activity was observed 3 hrs after the insult and prevented by systemic (S)-roscovitine administration. Our results show therefore that roscovitine protects in vivo neurons possibly through CDK5 dependent mechanisms.

Conclusions/Significance

Altogether, our data bring new evidences for the further development of pharmacological CDK inhibitors in stroke therapy.     

Insulin and glucagon share the same mechanism of neuroprotection in diabetic rats: role of glutamate

In patients with acute ischemic stroke, diabetes and hyperglycemia are associated with increased infarct size, more profound neurologic deficits and higher mortality. Notwithstanding extensive clinical and experimental data, treatment of stroke-associated hyperglycemia with insulin is controversial. In addition to hyperglycemia, diabetes and even early prediabetic insulin resistance are associated with increased levels of amino acids, including the neurotoxic glutamate, in the circulation. The pleiotropic metabolic effects of insulin include a reduction in the concentration of amino acids in the circulation. In this article, we show that in diabetic rats exposed to transient middle cerebral artery occlusion, a decrease of plasma glutamate by insulin or glucagon reduces CSF glutamate, improves brain histology, and preserves neurologic function. The neuroprotective effect of insulin and glucagon was similar, notwithstanding their opposite effects on blood glucose. The therapeutic window of both hormones overlapped with the short duration (~30 min) of elevated brain glutamate following brain trauma in rodents. Similar neuroprotective effects were found after administration of the glutamate scavenger oxaloacetate, which does not affect glucose metabolism. These data indicate that insulin and glucagon exert a neuroprotective effect within a very brief therapeutic window that correlates with their capacity to reduce glutamate, rather than by modifying glucose levels.     

All-Trans-Retinoic Acid Rescues Neurons After Global Ischemia by Attenuating Neuroinflammatory Reactions

Retinoic acid (RA) plays an important role in the developing mammalian nervous system. Based on this concept, some studies have demonstrated the beneficial effects of RA administration on neurogenesis in neuropathological diseases. Some investigations have revealed the anti-inflammatory effects of RA treatment in multiple systems, in addition to its role in neurogenesis. To date, however, the neuroprotective efficacy of RA after cerebral ischemia, especially in the context of its anti-inflammatory effects, has been poorly demonstrated. Additionally, to the best of our knowledge, experiments of the therapeutic efficacy of RA treatment in a transient global ischemic model in the Mongolian gerbil have been lacking worldwide. Here, we studied the neuroprotective effects and neurobehavioral outcomes of intraperitoneally administered all-trans-RA (ATRA; a synthetic form of RA) on brains with transient global ischemia that was induced with the bilateral common carotid artery occlusion and reperfusion (BCCAO/R) model in the gerbil. In order to identify whether these neuroprotective mechanisms were due to the anti-inflammatory effects of ATRA, in vivo hippocampal expression of proinflammatory cytokines including tissue necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) after ATRA injection and in vitro levels of release of nitric oxide, TNF-α and IL-6 from lipopolysaccharide (LPS)-stimulated BV2 microglial cells after ATRA treatment were evaluated. The results showed that ATRA can protect pyramidal neurons in the hippocampal CA1 region against BCCAO-induced neuronal apoptosis and significantly reduce the extent of astrocytosis and microglial activation. In addition, the ischemia-induced neurobehavioral changes were normalized by ATRA injection. Consistent with these phenotypic data, we observed the diminishing effects of ATRA treatment on the production of proinflammatory mediators (e.g., TNF-α and IL-6) in hippocampal homogenates and LPS-stimulated BV2 cells, and these effects were dose-dependent. These results suggest a beneficial role of ATRA in the attenuation of global cerebral ischemia due to its anti-inflammatory properties, resulting in, at least partly, the inhibition of microglial secretion of variable proinflammatory cytokines.     

Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction

Growing evidence from in vitro studies supports that valproic acid (VPA), an anti-convulsant and mood-stabilizing drug, has neuroprotective effects. The present study investigated whether VPA reduces brain damage and improves functional outcome in a transient focal cerebral ischemia model of rats. Subcutaneous injection of VPA (300 mg/kg) immediately after ischemia followed by repeated injections every 12 h, was found to markedly decrease infarct size and reduce ischemia-induced neurological deficit scores measured at 24 and 48 h after ischemic onset. VPA treatment also suppressed ischemia-induced neuronal caspase-3 activation in the cerebral cortex. VPA treatments resulted in a time-dependent increase in acetylated histone H3 levels in the cortex and striatum of both ipsilateral and contralateral brain hemispheres of middle cerebral artery occlusion (MCAO) rats, as well as in these brain areas of normal, non-surgical rats, supporting the in vitro finding that VPA is a histone deacetylase (HDAC) inhibitor. Similarly, heat shock protein 70 (HSP70) levels were time-dependently up-regulated by VPA in the cortex and striatum of both ipsilateral and contralateral sides of MCAO rats and in these brain areas of normal rats. Altogether, our results demonstrate that VPA is neuroprotective in the cerebral ischemia model and suggest that the protection mechanisms may involve HDAC inhibition and HSP induction.     

Apelin-13 Inhibits Apoptosis of Cortical Neurons Following Brain Ischemic Reperfusion Injury in a Transient Model of Focal Cerebral Ischemia

Stroke is the third leading cause of death world-wide, affecting 15 million people annually. Diminished blood supply to the brain cells is the main cause of damage following stroke. When focal ischemia occurs, the core of brain tissue influenced by reduced blood supply undergoes necrotic cell death. The adipocytokine Apelin is a peptide that was isolated from a bovine stomach for the first time. This peptide and its receptor are abundantly expressed in the nervous and cardiovascular systems. According to previous studies, Apelin-13 protects cardiomyocytes from ischemic injury and apoptosis. In addition, this peptide has neuroprotective effect on hippocampal and cultured mouse cortical neurons against NMDA receptor-mediated excitotoxicity as well as cortical neurons from ischemic injury. The present study was conducted to determine whether Apelin-13 inhibits apoptosis in the ischemic penumbra in transient focal cerebral ischemia. Focal cerebral ischemia was induced in male Wistar rats by 60 min middle cerebral artery occlusion (MCAO) using a filament method, followed by 23-h reperfusion. Saline as a vehicle and Apelin-13 at doses of 50 and 100 μg were injected intracerebro-ventriculary (ICV) at the beginning of ischemia. Apoptosis and neurological dysfunction were assessed 24-h after MCAO. Our results indicated that administration of Apelin-13 at doses of 50 and 100 μg ICV markedly reduced apoptosis by decreasing positive TUNEL cells (P < 0.001). In addition, Apelin-13 at doses of 100 μg significantly change neurological dysfunction (P < 0.05). Our findings demonstrate that treatment by Apelin-13 exerts its protective effects in ischemic models via blocking programmed cell-death. We suggest that Apelin-13 might be a promising therapeutic target for stroke, although more researches are necessary to take into account the potential therapeutic effects of Apelin-13 in stroke patients.     

Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist,Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats

Background

In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and Results

Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion

These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.     

Neuroprotective effect of choline succinate in rats with experimental chronic cerebral ischemia evaluated by cognitive ability tests

The neuroprotective effect of choline succinate was studied in rats with chronic cerebral ischemia induced by ligation of both common carotid arteries. Two approaches were used to evaluate the neuroprotective effect: cognitive ability tests (passive avoidance test and Morris water maze test) and determination of the brain content of N-acetylaspartate, a marker of functional neurons, by ¹H NMR spectroscopy in vivo. Choline succinate administration significantly improved memory and learning in ischemic rats and prevented the ischemia-induced decrease in the cerebral level of N-acetylaspartate. Thus, choline succinate demonstrated a neuroprotective effect in conditions of ischemic brain injury.     

Effect of anesthetics on efficiency of remote ischemic preconditioning

Remote ischemic preconditioning of hind limbs (RIPC) is an effective method for preventing brain injury resulting from ischemia. However, in numerous studies RIPC has been used on the background of administered anesthetics, which also could exhibit neuroprotective properties. Therefore, investigation of the signaling pathways triggered by RIPC and the effect of anesthetics is important. In this study, we explored the effect of anesthetics (chloral hydrate and Zoletil) on the ability of RIPC to protect the brain from injury caused by ischemia and reperfusion. We found that RIPC without anesthesia resulted in statistically significant decrease in neurological deficit 24 h after ischemia, but did not affect the volume of brain injury. Administration of chloral hydrate or Zoletil one day prior to brain ischemia produced a preconditioning effect by their own, decreasing the degree of neurological deficit and lowering the volume of infarct with the use of Zoletil. The protective effects observed after RIPC with chloral hydrate or Zoletil were similar to those observed when only the respective anesthetic was used. RIPC was accompanied by significant increase in the level of brain proteins associated with the induction of ischemic tolerance such as pGSK-3β, BDNF, and HSP70. However, Zoletil did not affect the level of these proteins 24 h after injection, and chloral hydrate caused increase of only pGSK-3β. We conclude that RIPC, chloral hydrate, and Zoletil produce a significant neuroprotective effect, but the simultaneous use of anesthetics with RIPC does not enhance the degree of neuroprotection.     

Trehalose Inhibits Protein Aggregation Caused by Transient Ischemic Insults Through Preservation of Proteasome Activity,Not via Induction of Autophagy

Protein aggregation has been proved to be a pathological basis accounting for neuronal death caused by either transient global ischemia or oxygen glucose deprivation (OGD), and inhibition of protein aggregation is emerging as a potential strategy of preventing brain damage. Trehalose was found to inhibit protein aggregation caused by neurodegenerative diseases via induction of autophagy, whereas its effect is still elusive on ischemia-induced protein aggregation. In this study, we investigated this issue by using rat model of transient global ischemia and SH-SY5Y model of OGD. We found that pretreatment with trehalose inhibited transient global ischemia-induced neuronal death in the hippocampus CA1 neurons and OGD-induced death in SH-SY5Y cells, which was associated with inhibition of the formation of ubiquitin-labeled protein aggregates and preservation of proteasome activity. In vitro study showed that the protection of trehalose against OGD-induced cell death and protein aggregation in SH-SY5Y cells was reversed when proteasome activity was inhibited by MG-132. Further studies revealed that trehalose prevented OGD-induced reduction of proteasome activity via suppression of both oxidative stress and endoplasmic reticulum stress. Particularly, our results showed that trehalose inhibited OGD-induced autophagy. Therefore, we demonstrated that proteasome dysfunction contributed to protein aggregation caused by ischemic insults and trehalose prevented protein aggregation via preservation of proteasome activity, not via induction of autophagy.