Ischemic preconditioning ameliorates excitotoxicity by shifting glutamate/gamma-aminobutyric acid release and biosynthesis

Excitotoxicity is recognized to play a major role in cerebral ischemia-induced cell death. The main goal of the present study was to define whether our model of ischemic preconditioning (IPC) promotes a shift from excitatory to inhibitory neurotransmission during the test ischemia to diminish metabolic demand during the reperfusion phase. We also determined whether gamma-aminobutyric acid (GABA) played a role in IPC-induced neuroprotection. Ten minutes of cerebral ischemia was produced by tightening the carotid ligatures bilaterally following hypotension. Samples of microdialysis perfusate, representing extracellular fluid, were analyzed for amino acid content by HPLC. IPC promoted a robust release of GABA after lethal ischemia compared with control rats. We also observed that the activity of glutamate decarboxylase (the predominant pathway of GABA synthesis in the brain) was higher in the IPC group compared with control and ischemic groups. Because GABAA receptor up-regulation has been shown to occur following IPC, and GABAA receptor activation has been implicated in neuroprotection against ischemic insults, we tested the hypothesis that GABAA or GABAB receptor activation was neuroprotective during ischemia or early reperfusion by using an in vitro model (organotypic hippocampal slice culture). Administration of the GABAB agonist baclofen during test ischemia and for 1 hr of reperfusion provided significant neuroprotection. We concluded that increased GABA release in preconditioned animals after ischemia might be one of the factors responsible for IPC neuroprotection. Specific activation of GABAB receptor contributes significantly to neuroprotection against ischemia in organotypic hippocampal slices.     

Brain ischemic preconditioning is abolished by antioxidant drugs but does not up-regulate superoxide dismutase and glutathion peroxidase

The present work examined the hypothesis that brain ischemic tolerance induced by ischemic preconditioning (IPC) is triggered by an initial oxidative stress and is associated with an increase in antioxidant enzyme activities as one end-effector of the neuroprotection. Wistar rats were preconditioned by a single 3-min occlusion of the middle cerebral artery. After a various duration of reperfusion (30 min, 24, 72 or 168 h), rats were subjected to a 60-min focal ischemia and sacrificed 24 h later. Cerebral infarcts were significantly reduced when performed during the 24- to 72-h time window after IPC. The pretreatment with the protein synthesis inhibitor, cycloheximide (1 mg/kg, i.p., 30 min prior to IPC), completely suppressed the neuroprotection. The free radical scavenger, dimethylthiourea (DMTU; 300 mg/kg, i.p., 30 min prior to IPC) and the antioxidant ebselen (10 mg/kg, oral cramming, 2 h before and 12 h after IPC) also abolished the IPC-induced protection of the brain. Nevertheless, IPC did not induce any delayed changes in antioxidant enzyme (superoxide dismutase, glutathion peroxidase) activities nor in the neuronal expression of Mn and Cu/Zn superoxide dismutase. These results indicate that an initial oxidative stress could be involved as a trigger of IPC, while antioxidant enzymes do not play a key role as end-effectors in such a neuroprotection.     

A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling,thereby reducing energy requirements during ischemia

Polyphenol resveratrol (RSV) has been associated with Silent Information Regulator T1 (SIRT1) and AMP‐activated protein kinase (AMPK) metabolic stress sensors and probably responds to the intracellular energy status. Our aim here was to investigate the neuroprotective effects of RSV and its association with SIRT1 and AMPK signaling in recurrent ischemia models. In this study, elderly male Wistar rats received a combination of two mild transient middle cerebral artery occlusions (tMCAOs) as an in vivo recurrent ischemic model. Primary cultured cortical neuronal cells subjected to combined oxygen–glucose deprivation (OGD) were used as an in vitro recurrent ischemic model. RSV administration significantly reduced infarct volumes, improved behavioral deficits and protected neuronal cells from cell death in recurrent ischemic stroke models in vivo and in vitro. RSV treatments significantly increased the intracellular NAD⁺/NADH ratio, AMPK and SIRT1 activities, decreased energy assumption and restored cell energy ATP level. SIRT1 and AMPK inhibitors and specific small interfering RNA (siRNA) for SIRT1 and AMPK significantly abrogated the neuroprotection induced by RSV. AMPK‐siRNA and inhibitor decreased SIRT1 activities; however, SIRT1‐siRNA and inhibitor had no impact on phospho‐AMPK (p‐AMPK) levels. These results indicated that the neuroprotective effects of RSV increased the intracellular NAD⁺/NADH ratio as well as AMPK and SIRT1 activities, thereby reducing energy ATP requirements during ischemia. SIRT1 is a downstream target of p‐AMPK signaling induced by RSV in the recurrent ischemic stroke model.     

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK‐3β) and cAMP response element‐binding protein (CREB) through phosphatidylinositol 3‐kinase (PI3‐K)‐dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four‐vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK‐3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3‐K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK‐3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3‐K, compromised the neuroprotective effect of resveratrol and decreased the level of p‐Akt, p‐GSK‐3β and p‐CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro‐survival states of Akt, GSK‐3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3‐K/Akt signaling pathway, subsequently downregulating expression of GSK‐3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.     

The effect of rapid preconditioning on the microglial, astrocytic and neuronal consequences of global cerebral ischemia

Previous studies indicated preconditioning of the brain with sublethal ischemic insults separated by many hours, protected tissues from a subsequent lethal insult. We recently reported neuroprotection by a rapid preconditioning paradigm where a sublethal ischemic insult preceded test ischemia by only 30 min. We hypothesize that neuroprotection caused by the rapid ischemic preconditioning (IPC) will result in lowered microglial, reactive astrocytes and increased normal neuronal cell counts. Wistar rats underwent normothermic (36.5–37 °C) global cerebral ischemia, produced by bilateral carotid artery ligation after lowering mean systemic blood pressure. The preconditioning ischemic insult lasted 2 min and was associated with a sufficient amount of time to provoke anoxic depolarization. After a 30-min reperfusion period, 10-min test ischemia was produced, and histopathology was assessed 3 and 7 days later. Normal neuronal cell counts for control rats at 3 days survival were significantly lower (by 58%) than in IPC animals. Although there was a trend toward protection in IPC rats at 7 days, the difference in normal neuronal cell count between the IPC and control groups was not significant. IPC rats at 3 days but not 7 days of survival showed a significantly lower microglial cell count (by 56%) than control rats. These results showed that the protection induced through IPC at 3 days of survival produced lower numbers of microglia, while maintaining normal neuronal cells. No significant differences between control and IPC groups were found in astrocytic cell count at any time of reperfusion in any region of the hippocampus studied. The beneficial effects of IPC may, therefore, involve anti-inflammatory processes that target microglial activation after cerebral ischemia. Received: 1 July 1998 / Revised: 30 September 1998 / Accepted: 21 October 1998     

Endogenous neuroprotection in the retina

Ischemic preconditioning (IPC) protects the rat retina against the injury that ordinarily follows severe ischemia. The retina is protected against the damage following severe ischemia for up to 72h after the application of IPC. However, there is no early preconditioning, i.e. protective effects starting within hours of preconditioning. The IPC stimulus consists of a brief, non-damaging period of ischemia. It results in complete preservation of retinal structure and function following ischemia, and is thus the most robust neuroprotection demonstrated in the retina to date. Release of adenosine, de novo protein synthesis, and mediators such as protein kinase C and K(+) ATP channels are required for IPC protection. Both the adenosine A1 and A2a receptors are involved. However, the molecular mechanisms for neuroprotection have not been completely described. It appears that both increased expression of protective proteins and decreased expression of pro-apoptotic proteins are involved. In addition, IPC prevents hypoperfusion following severe ischemia. Further study of the IPC phenomenon could lead to an enhanced understanding of the mechanisms of ischemic damage and its prevention in the retina.     

Cannabinoid receptor type 2 activation yields delayed tolerance to focal cerebral ischemia

We demonstrated in our previous research that pretreatment with electroacupuncture (EA) induces rapid (2h after EA) and delayed (24h after EA) tolerance to focal cerebral ischemia. We further elucidate the endocannabinoid and cannabinoid receptor type 1(CB1) involvment in the rapid ischemic tolerance induced by EA pretreatment. The present study aimed at investigating the involvement of the cannabinoid receptor type 2 (CB2) in the neuroprotection conferred by EA pretreatment. Focal cerebral ischemia was induced by middle cerebral artery occlusion for 120 min at 2h and 24h following EA pretreatment in male Sprague-Dawley rats, respectively. Cerebral ischemic injury was evaluated by neurobehavioral scores and infarction volume percentages 72 h after reperfusion in the presence or absence of AM251, a selective CB1 receptor antagonist, and AM630, a selective CB2 receptor antagonist. The expression of CB1 and CB2 receptor in the striatum of ischemic hemisphere was also evaluated. The rapid and delayed ischemic tolerance induced by EA pretreatment was respectively reversed by AM251 and AM630. CB2 receptor expression was up-regulated in the striatum of rat brains at 24h after EA stimuli. These results indicate that CB2 receptor contributed to the delayed neuroprotective effect whereas CB1 receptor to the rapid ischemic tolerance induced by EA pretreatment against focal cerebral ischemia in rats.     

Epsilon protein kinase C mediated ischemic tolerance requires activation of the extracellular regulated kinase pathway in the organotypic hippocampal slice.

Ischemic preconditioning (IPC) promotes brain tolerance against subsequent ischemic insults. Using the organotypic hippocampal slice culture, we conducted the present study to investigate (1) the role of adenosine A1 receptor (A1AR) activation in IPC induction, (2) whether epsilon protein kinase C (epsilonPKC) activation after IPC is mediated by the phosphoinositol pathway, and (3) whether epsilonPKC protection is mediated by the extracellular signal-regulated kinase (ERK) pathway. Our results demonstrate that activation of A1AR emulated IPC, whereas blockade of the A1AR during IPC diminished neuroprotection. The neuroprotection promoted by the A1AR was also reduced by the epsilonPKC antagonist. To determine whether epsilonPKC activation in IPC and A1AR preconditioning is mediated by activation of the phosphoinositol pathway, we incubated slices undergoing IPC or adenosine treatment with a phosphoinositol phospholipase C inhibitor. In both cases, preconditioning neuroprotection was significantly attenuated. To further characterize the subsequent signal transduction pathway that ensues after epsilonPKC activation, mitogen-activated protein kinase kinase was blocked during IPC and pharmacologic preconditioning (PPC) (with epsilonPKC, NMDA, or A1AR agonists). This treatment significantly attenuated IPC- and PPC-induced neuroprotection. In conclusion, we demonstrate that epsilonPKC activation after IPC/PPC is essential for neuroprotection against oxygen/glucose deprivation in organotypic slice cultures and that the ERK pathway is downstream to epsilonPKC.     

Electroacupuncture-induced neuroprotection against focal cerebral ischemia in the rat is mediated by adenosine A1 receptors

The activation of adenosine A1 receptors is important for protecting against ischemic brain injury and pretreatment with electroacu-puncture has been shown to mitigate ischemic brain insult. hTe aim of this study was to test whether the adenosine A1 receptor mediates electroacupuncture pretreatment-induced neuroprotection against ischemic brain injury. We ifrst performed 30 minutes of electroacu-puncture pretreatment at theBaihui acupoint (GV20), delivered with a current of 1 mA, a frequency of 2/15 Hz, and a depth of 1 mm. High-performance liquid chromatography found that adenosine triphosphate and adenosine levels peaked in the cerebral cortex at 15 minutes and 120 minutes atfer electroacupuncture pretreatment, respectively. We further examined the effect of 15 or 120 minutes electroacupuncture treatment on ischemic brain injury in a rat middle cerebral artery-occlusion model. We found that at 24 hours reper-fusion,120 minutes atfer electroacupuncture pretreatment, but not for 15 minutes, signiifcantly reduced behavioral deifcits and infarct volumes. Last, we demonstrated that the protective effect gained by 120 minutes atfer electroacupuncture treatment before ischemic injury was abolished by pretreatment with the A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 mg/kg, intraperitoneally). Our results suggest that pretreatment with electroacupuncture at theBaihui acupoint elicits protection against transient cerebral ischemiavia action at adenosine A1 receptors.     

Neuroimmune Response in Ischemic Preconditioning

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammtory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.     

Monocarboxylate transporter 4 plays a significant role in the neuroprotective mechanism of ischemic preconditioning in transient cerebral ischemia

Monocarboxylate transporters(MCTs), which carry monocarboxylates such as lactate across biological membranes, have been associated with cerebral ischemia/reperfusion process. In this study, we studied the effect of ischemic preconditioning(IPC) on MCT4 immunoreactivity after 5 minutes of transient cerebral ischemia in the gerbil. Animals were randomly designated to four groups(sham-operated group, ischemia only group, IPC + sham-operated group and IPC + ischemia group). A serious loss of neuron was found in the stratum pyramidale of the hippocampal CA1 region(CA1), not CA2/3, of the ischemia-only group at 5 days post-ischemia; however, in the IPC + ischemia groups, neurons in the stratum pyramidale of the CA1 were well protected. Weak MCT4 immunoreactivity was found in the stratum pyramidale of the CA1 in the sham-operated group. MCT4 immunoreactivity in the stratum pyramidale began to decrease at 2 days post-ischemia and was hardly detected at 5 days post-ischemia; at this time point, MCT4 immunoreactivity was newly expressed in astrocytes. In the IPC + sham-operated group, MCT4 immunoreactivity in the stratum pyramidale of the CA1 was increased compared with the sham-operated group, and, in the IPC + ischemia group, MCT4 immunoreactivity was also increased in the stratum pyramidale compared with the ischemia only group. Briefly, present findings show that IPC apparently protected CA1 pyramidal neurons and increased or maintained MCT4 expression in the stratum pyramidale of the CA1 after transient cerebral ischemia. Our findings suggest that MCT4 appears to play a significant role in the neuroprotective mechanism of IPC in the gerbil with transient cerebral ischemia.     

Resveratrol protects against global cerebral ischemic injury in gerbils

Increased oxidative stress has been implicated in the mechanisms of delayed neuronal cell death (DND) following cerebral ischemic insult. In this study, we investigated whether resveratrol, a polyphenolic antioxidant enriched in grape, may ameliorate ischemia-induced neuron cell death. Mongolian gerbils were divided into three groups, namely, sham control, ischemia and ischemia treated with resveratrol. Transient global cerebral ischemia was induced by occlusion of both common carotid arteries (CCA) for 5 min. Resveratrol was injected i.p. (30 mg/kg body weight), either during or shortly after CCA occlusion, and again at 24 h after ischemia. Cerebral blood flow was monitored before and during CCA occlusion using a laser Doppler flowmeter. Brain sections were immuno-stained for neurons, astrocytes and microglial cells. A time course study was also carried out to assess the bioavailability of resveratrol in serum, liver and brain using high performance liquid chromatography (HPLC). Morphometric measurements indicated extensive DND in the hippocampal CA1 region 4 days after ischemia and that neuron cell death was marked by the increase in reactive astrocytes and microglial cells. Administration of resveratrol, either during or after CCA occlusion, significantly (P<0.05) decreased DND as well as glial cell activation. Analysis of resveratrol after i.p. injection indicated its presence in serum, liver and brain with peak activity at 1, 4 and 4 h, respectively. This study demonstrated for the first time that resveratrol, a polyphenolic antioxidant, can cross the blood-brain barrier and exert protective effects against cerebral ischemic injury.     

Pretreated Oenan the Javanica extract increases anti-inflammatory cytokines,attenuates gliosis,and protects hippocampal neurons following transient global cerebral ischemia in gerbils

Recently, we have reported that Oenanthe javanica extract(OJE) displays strong neuroprotective effect against ischemic damage after transient global cerebral ischemia. However, neuroprotective mechanisms of OJE have not been fully identified. Thus, this study investigated the neuroprotection of OJE in the hippocampal CA1 area and its anti-inflammatory activity in gerbils subjected to 5 minutes of transient global cerebral ischemia. We treated the animals by intragastrical injection of OJE(100 and 200 mg/kg)once daily for 1 week prior to transient global cerebral ischemia. Neuroprotection of OJE was observed by immunohistochemistry for neuronal nuclear antigen and histofluorescence staining for Fluoro-Jade B. Im-munohistochemistry of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 was done for astrocytosis and microgliosis, respectively. To investigate the neuroprotective mechanisms of OJE,we performed immunohistochemistry of tumor necrosis factor-alpha and interleukin-2 for pro-inflamma-tory function and interleukin-4 and interleukin-13 for anti-inflammatory function. When we treated the animals by intragastrical administration of 200 mg/kg of OJE, hippocampal CA1 pyramidal neurons were protected from transient global cerebral ischemia and cerebral ischemia-induced gliosis was inhibited in the ischemic hippocampal CA1 area. We also found that interleukin-4 and -13 immunoreactivities were significantly increased in pyramidal neurons of the ischemic CA1 area after OJE pretreatment, and the increased immunoreactivities were sustained in the CA1 pyramidal neurons after transient global cere-bral ischemia. However, OJE pretreatment did not increase interleukin-2 and tumor necrosis factor-alpha immunoreactivities in the CA1 pyramidal neurons. Our findings suggest that pretreatment with OJE can protect neurons and attenuate gliosis from transient global cerebral ischemia via increasing expressions of interleukin-4 and -13. The experimental plan of this study was reviewed and approved by the Institutional Animal Care and Use Committee(IACUC) in Kangwon National University(approval No. KW-160802-1)on August 10, 2016.     

Ischemic preconditioning preserves mitochondrial function after global cerebral ischemia in rat hippocampus.

Ischemic tolerance in brain develops when sublethal ischemic insults occur before "lethal" cerebral ischemia. Two windows for the induction of tolerance by ischemic preconditioning (IPC) have been proposed: one that occurs within 1 hour after IPC, and another that occurs 1 or 2 days after IPC. The authors tested the hypotheses that IPC would reduce or prevent ischemia-induced mitochondrial dysfunction. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mm Hg) for 2 and 10 minutes, respectively. Nonsynaptosomal mitochondria were harvested 24 hours after the 10-minute "test" ischemic insult. No significant changes were observed in the oxygen consumption rates and activities for hippocampal mitochondrial complexes I to IV between the IPC and sham groups. Twenty-four hours of reperfusion after 10 minutes of global ischemia (without IPC) promoted significant decreases in the oxygen consumption rates in presence of substrates for complexes I and II compared with the IPC and sham groups. These data suggest that IPC protects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia.     

脑缺血预适应标志物的评估

缺血预适应是一种内源性神经保护机制,即短暂的、非致死缺血损伤保护后续的致死性 缺血打击。它通过短暂的缺血诱导固有的防御系统,引起对随后的严重缺血的耐受。预适应存在于多 种器官及生物,其中脑缺血预适应受到了广泛的关注。找到适合的标志物以表明预适应反应诱发的 缺血耐受是转化研究的重点。理想的标志物应易于获得,有较高的敏感性及特异性。本文将对可能 反映缺血预适应诱发的缺血耐受状态的血清及影像学标志物予以综述。     

Rapid tolerance to focal cerebral ischemia in rats is attenuated by adenosine A1 receptor antagonist.

Two types of ischemic tolerance in the brain, rapid and delayed, have been reported in terms of the interval between the conditioning and test insults. Although many reports showed that delayed-phase neuroprotection evoked by preconditioning is evident after 1 week or longer, there have been a few investigations about rapidly induced tolerance, and the reported neuroprotective effects become ambiguous 7 days after the insults. The authors examined whether this rapid ischemic tolerance exists after 7 days of reperfusion in a rat focal ischemic model, and investigated modulating effects of the adenosine A 1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine). Preconditioning with 30 minutes of middle cerebral artery occlusion reduced infarct volume 7 days after 180 minutes of subsequent focal ischemia given after 1-hour reperfusion. The rapid preconditioning also improved neurologic outcome. These beneficial effects were attenuated by pretreatment of 0.1 mg/kg DPCPX, which did not influence the infarct volume after conditioning (30 minutes) or test (180 minutes) ischemia when given alone. The results show that preconditioning with a brief focal ischemia induces rapid tolerance to a subsequent severe ischemic insult, the effect of which is still present after 7 days of reperfusion, and that the rapid ischemic tolerance is possibly mediated through an adenosine A 1 receptor-related mechanism.     

Neuroprotection of GST, an extract of traditional Chinese herb, against ischemic brain injury induced by transient brain ischemia and reperfusion in rat hippocampus

In this study, we investigated the effect of GST, an extract of Chinese traditional herb, on transient brain ischemia/reperfusion-induced neuronal cell death. Immunoblotting was used to detect the phosphorylation of MLK, JNK and c-jun. Transient (15 minutes) brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. GST was administrated to the SD rats 20 minutes before ischemia or 1 hour after ischemia. Our data showed that the pretreatment of GST could inhibit phosphorylation of MLK, JNK and c-jun. Moreover, GST showed potent neuroprotective effects on ischemic brain damage in vivo and administration of it 1 hour after ischemia also achieved the protective effects. These results indicate that GST has a prominent neuroprotection action against brain ischemic damage and provides a promising therapeutic approach for ischemic brain injury.     

Resveratrol protects against homocysteine‐induced cell damage via cell stress response in neuroblastoma cells

Recent findings underscore that some natural compounds are responsible for specific biochemical effects, i.e., the activation of redox‐sensitive intracellular pathways and modulation of different stress proteins, such as heat shock proteins and sirtuins. Resveratrol, a natural polyphenol widely present in plants, has been shown to display various beneficial effects, including neuroprotection, in several pathological conditions. In the present study, by using differentiated SH‐SY5Y neuroblastoma cells, we investigated the potential protective effects of resveratrol against homocysteine‐induced neurotoxicity. We observed that homocysteine (100 µM) decreased cell viability while at the same time significantly increasing intracellular reactive oxygen species and DNA fragmentation. Cell pretreatment with resveratrol concentrations ranging from 1 to 5 µM elicited protective effects through the reduction of oxidative stress and genotoxic damage. In addition, we observed that resveratrol produced significant changes in the expression of both Hsp70 and sirtuin 1 (SIRT1). After homocysteine treatment in the presence of resveratrol, SIRT1 protein was found abundantly not only in the cytosol but also in the nucleus, as demonstrated by confocal laser scanning microscopy. The results of this study suggest that resveratrol is a potential protective agent against homocysteine‐induced neurotoxicity and that beneficial effects are accompanied by changes in cell stress response. Taken together, these features contribute to our knowledge of underlying mechanisms involved in resveratrol‐induced cell survival. © 2014 Wiley Periodicals, Inc.     

Transforming growth factor-beta 1-mediated neuroprotection against excitotoxic injury in vivo.

Ischemic preconditioning is a phenomenon that describes how a sublethal ischemic insult can induce tolerance to subsequent ischemia. This phenomenon has been observed after focal or global ischemia in different animal models. However, the hypothesis that bacterial infection might lead to neuronal tolerance to injury has not been investigated. To mimic cerebral bacterial infection, we injected bacterial lipopolysaccharide (LPS) in the right dorsal hippocampus, followed 24 hours later by an excitotoxic lesion using kainic acid in the mouse model. Quantification of lesion size after cresyl violet counterstaining revealed that LPS pretreatment afforded neuroprotection to CA3 neurons against KA challenge. To investigate the events underlying this protection, we studied the cytokine profile induced after LPS injection. Interleukin (IL)-1 beta and transforming growth factor beta 1 (TGF-beta 1) were the main cytokines expressed at 24 hours after LPS injection. Because IL-1 beta has been described as deleterious in acute injury, we decided to investigate the function of TGF-beta 1. An adenovirus expressing a constitutively active form of TGF-beta 1 was injected intracerebrally 1 week before the induction of excitotoxic lesion, and neuronal protection was observed. To confirm the neuroprotective role of TGF-beta 1, the TGF-beta 1 adenovirus was replaced by recombinant human TGF-beta 1 protein and total neuroprotection was observed. Furthermore, the antibody-mediated blocking of TGF-beta 1 action prevented the protective effect of pretreatment with LPS. We have demonstrated in vivo that the cerebral tolerance phenomenon induced by LPS pretreatment is mediated by TGF-beta 1 cytokine.     

Aging blunts ischemic-preconditioning-induced neuroprotection following transient global ischemia in rats

The present study examines the hypothesis that aging defined by the 50% survival age compromises neuroprotection afforded by ischemic preconditioning (IPC). Sixty-four male F344 rats aged 4- and 24-months, respectively, were subjected to IPC, (3-min ischemia) or sham-surgery followed by 10-min (full) ischemia or sham-surgery 2 days later. There were 4 groups at each age: sham-surgery-sham-surgery (SS), preconditioning-sham-surgery (PS), preconditioning-ischemia (PI) and sham-surgery-ischemia (SI) groups. Assessments of histology and immunoreactivities of N-methyl-D-aspartic acid receptor 1 (NMDAr1) and caspase-3 active peptide (C3AP) in the hippocampal CA1 region were performed 8 days after full ischemia. The CA1 "living cell ratio" was greater in the aged SI group than in the young SI group (32+/-6% vs. 17+/-5%, p<0.05), whereas the degree of protection against full ischemia afforded by IPC was reduced in the aged compared with the young (53+/-17% vs. 241+/-25%, P<0.0001). The basal level of NMDAr1 immunofluorescence was significantly higher in young animals, while the numbers of C3AP-positive cells were greater in all three aged ischemic groups as compared to respective young groups (p<0.01, p=0.055 and p<0.05). A fourth method of assessing cell damage using Fluoro Jade C labeled degenerating neurons that were also intensively eosinophilic. Counts of Fluoro Jade C-positive cells were higher in the young SI group than in the aged SI group (P<0.05), suggesting that mechanisms of ischemic cell death may change with aging. In conclusion, aging alters mechanisms of ischemic cell death in CA1 neurons and ischemic tolerance mechanisms are blunted by aging.