506. Increased activity of small conductance calcium-activated postassium channels decrease hippocampal neuronal damage following global cerebral ischemia in adult mouse
P.S. Herson1, D. Allen1,2, T. Nakano1, J.P. Adelman2 and M. Kuroiwa1
1Department of Anesthesiology and Peri-Operative Medicine; 2Vollum Institute, Oregon Health & Sciences University, Portland, Oregon, USA
Objective: We recently demonstrated that small conductance calcium-activated potassium (SK) channels function as endogenous negative modulators of NMDA-receptors in hippocampal CA1 neurons,
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leading us to hypothesize that treatments resulting in increased SK channel activity would protect neurons against ischemia-induced damage by decreasing NMDA receptor mediated excitotoxicity. Therefore, we examined the survival of CA1 neurons following cardiac arrest and cardiopulmonary resuscitation (CA/CPR)-induced ischemia in male mice in which SK activity has been increased either genetically (SK2-over expression; SK2 OE) or pharmacologically with 1-EBIO. In addition, we examined the SK2 knockout mice (SK2 KO).
Methods: C57BL/6 WT, SK2 KO and SK2 OE mice underwent 8 mins. CA/CPR, as described previously.
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WT male mice were administered the SK channel agonist, 1-EBIO (16 mg/kg, intraperotineal (ip) injection), 30 min. prior to CA/CPR and given a subsequent boosting injection 6 h. after resuscitation. Three days after CA/CPR, brains were removed, embedded in paraffin and 6 μm coronal sections serially cut. Sections were stained with hematoxylin and eosin (H&E) for analysis of damaged neurons. The entire length of the CA1 region of the hippocampus was counted in three levels (100 μm apart) beginning from −1.5 mm bregma and the percentage of damaged neurons calculated for each brain. The investigator who analyzed neuronal damage was blinded to the treatment group. A separate group of mice received a femoral artery catheter for continuous monitoring of blood pressure and obtaining blood samples at 10 mins prior to arrest and at 30 mins post-resuscitation for measurement of arterial blood gases, pH, sodium, potassium, lactate and glucose levels.
Results: The absence of SK2 channels (SK2 KO) appears to exacerbate CA1 neuronal damage following CA/CPR, increasing neuronal death by ∼30% (from 36.9%±6.5% (n = 10) for control, to 50.6%±7.3% (n = 8)). In contrast, SK2-OE mice were significantly protected following CA/CPR, decreasing neuronal damage by ∼70%, to 10.5%±5.2% (n = 7; P<0.05). Similarly, the administration of 1-EBIO significantly reduced CA1 neuronal damage, from 62.4%±8.6% (n = 9) for vehicle to 33.9%±8.9% (n = 9; P<0.05) for 1-EBIO treated mice. Physiological parameters, including arterial blood gases, pH, sodium, potassium, lactate and blood glucose was not significantly affected by 1-EBIO administration. CPR time and epinephrine dose used for resuscitation in each group was not different.
Conclusions: The current data indicates that genetic over-expression or 1-EBIO-mediated pharmacological enhancement of synaptic SK2 channel activity increases CA1 neuron survival following ischemic insult. Therefore, SK2 channels represent a new therapeutic target for neuroprotection following cerebral ischemia.
614. NR2A subunit plays a crucial role on ischemic tolerance in cultured cortical neuron
Y. Terasaki1, T. Sasaki1, Y. Yagita2, S. Okazaki2, Y. Sugiyama2, N. Oyama1, E. Omura-Matsuoka1, S. Sakoda2 and K. Kitagawa2
1Division of Stroke Center, Department of Internal Medicine; 2Department of Neurology, Osaka University Graduate School of Medicine, Suita, Japan
Objectives: Based on the calcium-glutamate hypothesis, it has been emphasized that N-methyl D-aspartate (NMDA) glutamate receptor plays an important role of calcium influx to neuron. Clinically, the antagonist of glutamate receptor such as MK-801 proved ineffective. Recently, the difference between the subtypes of NMDA glutamate receptor was reported to affect downstream signal transduction and cell survival. Neuroprotective effect by ischemic tolerance has been reported to associate with up-regulation or down-regulation of various gene expressions. However the detailed mechanism is not fully clarified. The activation of a transcriptional factor, cyclic AMP response element binging protein (CREB), signaling contributes to a neuroprotective effect and phosphorylation of CREB serine 133 has been reported in ischemic tolerance. Within last decade, it has been reported that NR2A, a subunit of synaptic NMDA glutamate receptor, stimulated CREB function. We examined CRE transcriptional activity and the association with the subtypes of NMDA receptor and CREB phosphorylation in ischemic tolerance.
Methods: Primary cortical neuronal cultures were prepared from 16-day-old Wistar rat embryos and used 10 to 12 days in vitro. To examine whether ischemic tolerance was induced, cells were exposed to lethal 180 min oxygen-glucose deprivation (OGD) 1 to 48 h after sublethal 45 min OGD. Neuronal death was quantified by measurement of lactate dehydrogenase (LDH) released by dead neurons into the bathing medium. The expression of transcriptional factor, total CREB and phosphorylated CREB was assessed by Western blotting. To evaluate cyclic AMP response element (CRE) gene expression, cells were transfected by adenovirus-CRE reporter and the luciferase activities were assayed after stimulation using a Dual-Glo luciferase assay system. To elucidate the role of CREB phosphorylation in ischemic tolerance, inhibition of that was performed with an adenovirus-dominant negative CREB mutant in which the phosphorylation site at serine 133 was changed to alanine (CREB-S133A). To evaluate whether NR2A was associated with induction of ischemic tolerance, NR2A-specific antagonist NVP-AAM077 was added to the bath medium between sublethal and lethal OGD. Additionally, we examined whether the treatment of bucuculline to block GABA receptors and stimulate NR2A induced ischemic tolerance. Whether cross tolerance was induced, glutamate (50 μmol/L) or hydrogen peroxide (50 μmol/L) was administered 24 h after sublethal OGD.
Results: Ischemic tolerance was induced to be exposed to lethal 180 min OGD 24 h after sublethal 45 min OGD. The protective efficacy of preconditioning persisted for 12 to 48 h after sublethal OGD. After sublethal OGD p-CREB protein expression and CRE luciferase activity were upregulated. When cells were transfected by CREB-S133A, CRE luciferase activity was not upregulated and ischemic tolerance was lost. Next, we examined whether NR2A was associated with ischemic tolerance. To add NVP-AAM077 to the bath medium, the effectiveness of ischemic tolerance was lost. The administration of bucuculline enhanced CRE luciferase activity and induced ischemic tolerance. This neuroprotective effect of preconditioning could be also seen in other lethal injuries such as glutamate and hydrogen peroxide.
Conclusions: To obtain ischemic tolerance, it is important that neuroprotective signals through NR2A subunit of NMDA glutamate receptor induce phosphorylation of CREB protein and CRE transcriptional activity.
864. Calpain-dependent cleavage of eukaryotic translation initiation factor 4 G mediates translation arrest and cell death after ischemic injury
P. Vosler, C. Brennan and J. Chen
Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
Objectives: Focal ischemia results in uniform translation arrest in all ischemia-affected neurons. Importantly, the area of translation arrest is consistent throughout reperfusion and nearly completely coincides with total infarct area. Thus, translation arrest is a robust predictor of brain infarct. Our overarching hypothesis is that alleviation of translation arrest will confer neuroprotection. Previous in vivo studies have shown there is a decrease in the protein levels of the scaffolding protein responsible for transporting mRNA to the ribosome, eukaryotic initiation factor (eIF) 4G. It has been suggested that eIF4G is cleaved by the protease calpain. Our experiments attempt to determine if prevention of eIF4G cleavage is sufficient to restore translation and increase neuronal viability following ischemia.
Methods: The in vitro ischemia model oxygen glucose deprivation (OGD) was used on rat primary cortical neurons. Alamar blue and Hoechst staining measured cell viability and translation was measured with TCA precipitation. Calpain activity was confirmed using calpain activity assay and a-spectrin cleavage. In vitro cleavage assays were performed with recombinant proteins. Lentiviral vectors were used to overexpress eIF4G constructs.
Results: Sublethal OGD resulted in a significant decrease in translation immediately after OGD with complete recovery by 12 to 24 h of reperfusion. In contrast, lethal OGD resulted in a similar initial inhibition of translation but with recovery to only 50% of control values. Translation recovery was correlated with cell viability, as sublethal OGD did not result in decreased viability and lethal OGD resulted in approximately 50% viability. The levels of translation initiation factors were unchanged except for eIF4G, which progressively decreased from 2 to 24 h reperfusion. Further examination demonstrated that eIF4G was cleaved in a caspase-independent manner by calpain, as overexpression of the endogenous calpain inhibitor, calpastatin, inhibited eIF4G cleavage, restored translation, and increased neuronal viability above vector-treated control following OGD. To definitively establish eIF4GI as a calpain substrate, an in vitro assay was performed. Activation of recombinant calpain with Ca2+ progressively cleaved recombinant eIF4GI over 30 min-an effect that was blocked by calpain-specific inhibitors. We next determined if neuronal death following ischemia was directly related to maintenance of critical levels of eIF4G. Overexpression of HA-eIF4G, but not a mutant eIF4G lacking the cap-binding protein binding site, increased translation and viability above eGFP-vector controls. This suggests that increased neuronal viability due to eIF4G overexpression is due to the restoration of cap-dependent translation.
Conclusions: This work describes the novel mechanism of calpain-mediated cleavage of eIF4G in ischemia-induced translation arrest and neuronal death following in vitro ischemia. Our findings suggest that the level and integrity of eIF4G are crucial to the maintenance of translation and neuronal viability after ischemia. Future work is aimed at determining the biological significance of eIF4G in terms of the types of mRNA translated following ischemia. Using luciferase constructs we are attempting to determine if the 5′UTRs of various viral and cellular internal ribosomal entry sequences (IRES) are preferentially translated following ischemia. Understanding the pathophysiological consequences of eIF4G cleavage and translation arrest may lead to novel therapeutic strategies to ameliorate neuronal death following stroke.
250. Effects of transcranial near-infrared laser irradiation on cerebral blood flow in mice
H. Nawashiro1, Y. Uozumi1, S. Sato2, S. Kawauchi3, K. Shima1 and M. Kikuchi3
1Neurosurgery, National Defense Medical College; 2Biomedical Information Sciences, National Defense Medical College Research Institute; 3Medical Engineering, National Defense Medical College, Tokorozawa, Japan
Objectives: Photobiostimulation effects of near-infrared (NIR) laser irradiation have been known for almost forty years.
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Recently it has been reported that NIR laser irradiation is effective in cerebral ischemia in vivo and clinically.
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We examined the effect of 808 nm laser diode irradiation on CBF in mice. Furthermore, we examined the role of nitric oxide and a neurotransmitter, glutamate to regulate CBF in this animal model. The potential of NIR laser irradiation in the treatment of cerebral ischemia was also investigated.
Methods: Male C57BL/6J mice 9- to 11-weeks old and weighing 23 to 27 g (CLEA Japan, Inc, Tokyo, Japan) were used in the study. Under general and local anesthesia, an 808 nm CW diode laser (B&W Tek, Inc, Newark, DE) was applied to the left hemisphere transcranially. To determine the appropriate power density (PD) of NIR laser irradiation, we irradiated the laser with three different PD (0.8 W/cm2; n = 6, 1.6 W/cm2; n = 9, 3.2 W/cm2; n = 6), and measured the PD using a laser power meter with photodiode head (Laserstar; Ophir Optronics Ltd, Jerusalem, Israel) before and after every irradiation. CBF was measured in the cortex with a non-invasive and non-contact laser Doppler blood perfusion imager (PeriScan PIM II, PeriMed, Stockholm, Sweden). We measured directly nitric oxide in the brain tissue during NIR laser irradiation by an amperometric nitric oxide (NO)-selective electrode (IMN-111, Inter Medical, Nagoya, Japan). Furthermore, in Ng-nitro-L-arginie methyl ester hydrochloride (L-NAME) treated mice NO was also measured during NIR laser irradiation (n = 4). To gain insight into potential mechanisms linking neurotransmission with CBF, effects of a non-competitive N-methyl-D-aspartate receptor blocker, MK-801 was investigated. To confirm the effect of pretreatment by NIR laser irradiation, we conducted the 1.6 W/cm2 NIR laser irradiation to the left hemisphere transcranially for 30 min before bilateral common carotid artery occlusion (BCCAO) (n = 13). The control mice (n = 13) were also subjected to 15 min BCCAO without pretreatment by NIR laser irradiation.
Results: Transcranial NIR laser irradiation (1.6 W/cm2 for 15 to 45 min, wavelength, 808 nm) increased local CBF by 30% compared to control value in mice. NIR laser irradiation also provoked a significant increase in cerebral NO concentration. Mice that received the broad-spectrum NO synthase (NOS) inhibitor, L-NAME did not show any CBF increase by NIR laser irradiation. Mice administered MK-801 showed immediate increase but loss of delayed increase in local CBF by NIR laser irradiation. Pretreatment by NIR laser irradiation improved residual CBF following bilateral carotid occlusion in mice.
Conclusions: Our data suggest that targeted increase of CBF is available by NIR laser irradiation and it is concerned in NOS activity and NO concentration. Besides, NIR laser irradiation may have a protective effect for transient ischemia.
820. Mitochondrial dysfunction induced by focal cerebral ischemia in the TNFα-transgenic rat
J. Pandya1, P. Sullivan1 and L. Pettigrew2,3
1Spinal Cord & Brain Injury Research Center; 2Stroke Program, Sanders-Brown Center on Aging, University of Kentucky; 3Department of Veterans Affairs Medical Center, Lexington, Kentucky, USA
Objectives: Tumor necrosis factor-alpha (TNFα) induces neurodegeneration in ischemic brain through cytokine-receptor mediated signalling pathways. Current evidence suggests that neuronal mitochondria stressed by ischemia may contribute synergistically to TNFα-mediated cell death. We tested the hypothesis that increased synthesis of TNFα in ischemic brain will promote oxidative stress through mitochondrial dysfunction.
Methods: TNFα-transgenic (TNFα-Tg) rats were constructed to overexpress the murine TNFα gene, resulting in elevated brain levels of active TNFα protein.
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Both TNFα-Tg rats and non-Tg littermates of male sex underwent reversible middle cerebral artery occlusion (MCAO) for 1 h. Mitochondria were isolated from ischemic cortex (IC) sampled after 3 h of reperfusion. Production of reactive oxygen species (ROS) in mitochondria was measured with 2,7-dichlorodihydrofluorescein.
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Results: The mean ROS level in cortical mitochondria sampled from non-ischemic TNFα-Tg brain (n = 7) was 349±26.7 (s.e.m.) relative fluorescence units, compared to 277.4±20 in non-Tg brain (n = 8; P = 0.05; unpaired t-test). After MCAO, the ROS level peaked within IC in TNFα-Tg brain (518.3±41.9) and was significantly greater than in unaffected cortex sampled from the same transgenic animals (385.3±51.9; P = 0.05) or from non-Tg littermates (293.8±15.1; P = 0.003; Fisher's test; n = 6 per group for all comparisons). The mean ROS level measured within IC in TNFα-Tg brain also exceeded that in ischemic brain of non-Tg rats (393.3±50) by a margin that approached statistical significance (P = 0.06).
Conclusions: Our results show that ROS is produced at higher basal levels in neural mitochondria isolated from TNFα-Tg rats than in non-Tg littermates, suggesting that elevated TNFα protein induces free radical synthesis even without physiological stress. After MCAO, we found that ROS synthesis was further amplified in ischemic brain sampled from TNFα-Tg animals. We conclude that ischemic stress and synthesis of inflammatory cytokines, such as may occur during post-ischemic reperfusion in human brain, will synergistically augment ROS production to promote neuronal death.
Grant support: NIH/NINDS NS047395 and Merit Review Award from the Veterans Administration (LCP).
278. Salt-induced kinase 1 regulated histone acetylation in primary neurons and its role in androgen-mediated neuroprotection in experimental stroke
J. Cheng, P. Herson, W. Zhang and P. Hurn
Oregon Health and Science University, Portland, Oregon, USA
Androgens alter experimental stroke outcomes in a dose- and androgen receptor-dependent manner. At physiological doses, both testosterone and its nonaromatizable metabolite dihyrdotestosteron (DHT) reduce infarct sizes after focal cerebral ischemia, and the protection is blocked by flutamide, a specific androgen receptor antagonist. Using microarray, we have previously shown that salt-induced kinase 1 (SIK-1) is induced by DHT in penumbral tissue after middle cerebral artery occlusion (MCAO). SIK1 belongs to the sucrose nonfermenting-1 protein kinase/AMP-activated protein kinase family and encodes a Ser/Thr kinase. In C. elegans, the homology of SIK-1 is believed to function as a histone deacetylase (HDAC) kinase, thus inhibiting HDAC activities and enhancing histone acetylation in neurons. The present study determined if SIK-1 is an important mediator of androgen neuroprotection in experimental stroke and acts by modulating histone acetylation. First, we confirmed by quantitative real-time PCR (qPCR) that SIK-1 was induced in the cortical and striatal penumbra of castrated male rats treated with DHT (15 mg/implant) at 6 and 12 h following MCAO, and SIK1 induction was lost with concomitant administration of flutamide (15 mg/pellet). To examine the involvement of SIK-1 in neuronal pathophysiology after ischemia, we constructed a lentivirus expressing small interference RNA (siRNA) with >70% knockdown efficiency on murine SIK-1 mRNA. Lentivirus-mediated SIK-1 knockdown significantly exacerbated neuronal cell death induced by oxygen and glucose deprivation (OGD) as determined by MTT assay and lactate dehydrogenase (LDH) release. At 22 h reoxygenation following 2.5 h OGD, MTT levels were decreased, and LDH release from damaged cells was increased in neurons infected with lentiviruses expressing SIK1 siRNA (MTT: 42%±7%; LDH: 65%±11%, n = 4) as compared to control neurons without lentiviral infection (MTT: 68%±5%; LDH: 54%±11%) or neurons infected with empty virus (MTT: 78%±9%; LDH: 54%±11%) or virus expressing unfuctional siRNA (MTT: 86%±10%; LDH: 45%±9%). Consistent with the hypothesis that SIK-1 functions as a HDAC kinase and promotes histone acetylation, SIK-1 knockdown resulted in a decrease in baseline histone acetylation in primary neurons. However, HDAC inhibitor trichostatin A (TSA)-enhanced baseline histone acetylation level was not attenuated by lentivirus-delivered siRNA against SIK-1. In contrast, SIK-1 knockdown blunted TSA-elevated histone acetylation at 6 h reoxygenation following 2.5 h OGD and subsequently abolished the neuroprotection of TSA at 22 h reoxygenation. Our results suggest that DHT-induced post-ischemic expression of SIK-1, a novel neuroprotective gene in vitro, may contribute to androgen's neuroprotective properties at physiologically relevant levels and may act as an endogenous HDAC kinase.
