SB 234551 selective ET(A) receptor antagonism: perfusion/diffusion MRI used to define treatable stroke model, time to treatment and mechanism of protection

Mismatches between tissue perfusion-weighted imaging (PWI; an index of blood flow deficit) and cellular diffusion-weighted imaging (DWI; an index of tissue injury) provide information on potentially salvageable penumbra tissue in focal stroke and can identify “treatable” stroke patients. The present pre-clinical studies were conducted to: a.) Determine PWI (using perfusion delay) and DWI measurements in two experimental stroke models, b.) Utilize these measurements to characterize selective ET_A receptor antagonism (i.e., determine efficacy, time-to-treatment and susceptibility to treatment in the different stroke models), and c.) Determine if increasing the reduced blood flow following a stroke is a mechanism of protection. Permanent middle cerebral artery occlusion (MCAO) or sham surgeries were produced in Sprague Dawley rats (SD; proximal MCAO; hypothesized to be a model of slowly evolving brain injury with a significant penumbra) and in spontaneously hypertensive rats (SHR; distal MCAO; hypothesized to be a model of rapidly evolving brain injury with little penumbra). Infusions of vehicle or SB 234551 (3, 10, or 30 µg/kg/min) were initiated at 0, 75, and/or 180 min post-surgery and maintained for the remainder of 24 h post-surgery. Hyper-intense areas of perfusion delay (PWI) in the forebrain were measured using Gadolinium (Gd) bolus contrast. DWI hyper-intense areas were also measured, and the degree of forebrain DWI-PWI mismatch was determined. Region specific analyses (ROI) were also conducted in the core ischemic and low perfusion/penumbra areas to provide indices of perfusion and changes in the degree of tissue perfusion due to SB 234551 treatment. At 24 h post-surgery, final infarct volume was measured by DWI and by staining forebrain slices. Following SD proximal MCAO, there was a significant mismatch in the ischemic forebrain PWI compared to DWI (PWI > DWI) at 60 min which was maintained up to 150 min (all p < 0.05). By 24 h post-stroke, infarct volume was identical to the area of early perfusion deficit/PWI, suggesting a slow progression of infarct development that expanded into the significant, earlier cortical penumbra (i.e., model with salvageable tissue with potential for intervention). When SB 234551 was administered within the period of peak mismatch (i.e., at 75 min post-stroke), SB 234551 provided significant dose-related reductions in cortical (penumbral) progression to infarction (p < 0.05). Cortical protection was related to an increased/normalization of the stroke-induced decrease in tissue perfusion in cortical penumbra areas (p < 0.05). No SB 234551-induced changes in reduced tissue perfusion were observed in the striatum core ischemic area. Also, when SB-234551 was administered beyond the time of mismatch, no effect on cortical penumbra progression to infarct was observed. In comparison and strikingly different, following SHR distal MCAO there was no mismatch between PWI and DWI (PWI = DWI) as early as 60 min post-stroke, with this early change in SHR DWI being identical to the final infarct volume at 24 h, suggesting a rapidly occurring brain injury with little cortical penumbra (i.e., model with little salvageable tissue or potential for intervention). In distal MCAO, SB 234551 administered immediately at the time of stroke did not have any effect on infarct volume in SHR. These data demonstrate that selective blockade of ET_A receptors is protective following proximal MCAO in SD (i.e. a model similar to “treatable” clinical patients). The protective mechanism appears to be due to enhanced collateral blood flow and salvage of penumbra. Therefore, the use of PWI-DWI mismatch signatures can identify treatable stroke models characterized by a salvageable penumbra and can define appropriate time to treatment protocols. In addition, tissue perfusion information obtained under these conditions might clarify mechanism of protection in the evaluation of protective compounds for focal stroke.     

Nicotinamide Prevents NAD+ Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia: NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons

Neurons require large amounts of energy to support their survival and function, and are therefore susceptible to excitotoxicity, a form of cell death involving bioenergetic stress that may occur in several neurological disorders including stroke and Alzheimer’s disease. Here we studied the roles of NAD⁺ bioenergetic state, and the NAD⁺-dependent enzymes SIRT1 and PARP-1, in excitotoxic neuronal death in cultured neurons and in a mouse model of focal ischemic stroke. Excitotoxic activation of NMDA receptors induced a rapid decrease of cellular NAD(P)H levels and mitochondrial membrane potential. Decreased NAD⁺ levels and poly (ADP-ribose) polymer (PAR) accumulation in nuclei were relatively early events (<4 h) that preceded the appearance of propidium iodide- and TUNEL-positive cells (markers of necrotic cell death and DNA strand breakage, respectively) which became evident by 6 h. Nicotinamide, an NAD⁺ precursor and an inhibitor of SIRT1 and PARP1, inhibited SIRT1 deacetylase activity without affecting SIRT1 protein levels. NAD⁺ levels were preserved and PAR accumulation and neuronal death induced by excitotoxic insults were attenuated in nicotinamide-treated cells. Treatment of neurons with the SIRT1 activator resveratrol did not protect them from glutamate/NMDA-induced NAD⁺ depletion and death. In a mouse model of focal cerebral ischemic stroke, NAD⁺ levels were decreased in both the contralateral and ipsilateral cortex 6 h after the onset of ischemia. Stroke resulted in dynamic changes of SIRT1 protein and activity levels which varied among brain regions. Administration of nicotinamide (200 mg/kg, i.p.) up to 1 h after the onset of ischemia elevated brain NAD⁺ levels and reduced ischemic infarct size. Our findings demonstrate that the NAD⁺ bioenergetic state is critical in determining whether neurons live or die in excitotoxic and ischemic conditions, and suggest a potential therapeutic benefit in stroke of agents that preserve cellular NAD⁺ levels. Our data further suggest that, SIRT1 is linked to bioenergetic state and stress responses in neurons, and that under conditions of reduced cellular energy levels SIRT1 enzyme activity may consume sufficient NAD⁺ to nullify any cell survival-promoting effects of its deacetylase action on protein substrates.     

Migration and homing of bone-marrow mononuclear cells in chronic ischemic stroke after intra-arterial injection

Cell-based treatments have been considered a promising therapy for neurological diseases. However, currently there are no clinically available methods to monitor whether the transplanted cells reach and remain in the brain. In this study we investigated the feasibility of detecting the distribution and homing of autologous bone-marrow mononuclear cells (BMMCs) labeled with Technetium-99 m (^99mTc) in a cell-based therapy clinical study for chronic ischemic stroke. Six male patients (ages 24-65 years) with ischemic cerebral infarcts within the middle cerebral artery (MCA) between 59 and 82 days were included. Cell dose ranged from 1.25 × 10⁸ to 5 × 10⁸. Approximately 2 × 10⁷ cells were labeled with ^99mTc and intra-arterially delivered together with the unlabeled cells via a catheter navigated to the MCA. None of the patients showed any complications on the 120-day follow-up. Whole body scintigraphies indicated cell homing in the brain of all patients at 2 h, while the remaining uptake was mainly distributed to liver, lungs, spleen, kidneys and bladder. Moreover, quantification of uptake in Single-Photon Emission Computed Tomography (SPECT) at 2 h showed preferential accumulation of radioactivity in the hemisphere affected by the ischemic infarct in all patients. However, at 24 h homing could only distinguished in the brains of 2 patients, while in all patients uptake was still seen in the other organs. Taken together, these results indicate that labeling of BMMCs with ^99mTc is a safe and feasible technique that allows monitoring the migration and engraftment of intra-arterially transplanted cells for at least 24 h.     

Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia.

We investigated the temporal threshold for focal cerebral infarction in the spontaneously hypertensive rat. The right middle cerebral artery and common carotid artery were occluded for 0, 1, 2, 3, 4, or 24 hours, and all the animals were sacrificed 24 hours after the onset of ischemia. Cortical infarct volumes and edema volumes were quantified in serial frozen sections of hematoxylin and eosin-stained tissue using image analysis. Upon occlusion, blood flow in the core of the ischemic zone, measured with laser-Doppler flowmetry, fell to a mean +/- standard deviation of 21 +/- 7% of the preocclusion baseline value (n = 26). During the first hour of ischemia, blood flow in the densely ischemic zone rose to 27 +/- 8% of baseline (n = 25). Release of the middle cerebral artery and common carotid artery occlusions rapidly restored cortical blood flow to 213 +/- 83% of baseline (n = 21). Focal ischemia of 1 hour's duration caused little or no infarction, while ischemic intervals of 2 and 3 hours produced successively larger volumes of infarcted cortex. Ischemic intervals of 3-4 hours' duration followed by approximately 20 hours of recirculation yielded infarct volumes that were not significantly different from those after 24 hours of permanent focal ischemia. The results indicate that 3-4 hours of focal cerebral ischemia in this rat model is sufficient to attain maximal infarction and suggest that recirculation or pharmacological interventions after this time will provide little benefit.     

Morphological and neuro-behavioral parallels in the rat model of stroke

Middle cerebral artery occlusion (MCAO) is widely used as a rat model of focal brain ischemia. Evaluation of brain damage often includes the morphological analysis of the injury area, MRI, and various scales which depend on functional tests, commonly known as neurological severity score (NSS). We determined the optimal number of NSS tests and assessed their capacity for non-invasive evaluation of brain ischemic injury in the rat MCAO model. 275 male Sprague-Dawley rats were randomly divided into five groups, given either permanent (p) MCAO or transient (t) MCAO using an uncoated 4-0 monofilament catheter or a silicone-coated monofilament. The rats’ neurological status was examined before and at 1 and 24 h following MCAO. The size of brain injury was then measured histologically and the extent of right cerebral hemisphere edema was calculated. We established a correlation between these tests and morphological data for brain injury. Adjusted R² of the prediction of total histology score was 0.7. The Hosmer-Lemeshow p-value of this model was 0.812 for total brain histology. For the brain edema the adjusted R² of the prediction model was 0.48. The Hosmer-Lemeshow p-value of this model was 0.558 for brain edema. Our methods of estimating infarct size produces reliable and well correlated results at 24 h and demonstrates to be an easy and quick way to assess infarct size soon after ischemic injury has occurred. The described method for neurological assessment could ultimately aid in assessing various treatment modalities in the early hours following stroke.     

Glutamate receptor antagonists modulate heat shock protein response in focal brain ischemia

Abstract

Neurons and glia reacting to ischemic injury exhibit delayed expression of heat shock proteins (HSPs). We tested the hypothesis that glutamate receptor antagonists alter neuronal and glial activation during focal cerebral ischemia, as shown by spatio–temporal changes in HSP immunoreactivity. Rats underwent focal ischemia by permanent occlusion of the middle cerebral artery. All animals were pre-treated with NBQX (30 mg kg^-1), a competitive antagonist of the AMPA/kainate receptor, or CGS-19755 (10 mg kg^-1), a competitive NMDA receptor antagonist, and euthanatized after 6 or 24 h of ischemia to demonstrate regional immunoreactivity of HSP-72 or 32 in brain. Neurons immunolabeled for HSP-72 appeared in the penumbral region adjacent to the infarct at 24 h and increased in number and distribution after pretreatment with NBQX or CGS-19755. Immunolabeling for HSP-32 revealed that pre-treatment with CGS19755 caused ramified glia to infiltrate the ischemic cortex at 6 h, a pattern that was not seen in ischemic controls until 24 h. Blockade of the NMDA or AMPA/kainate receptor modulates cellular stress responses in both neurons and glia within the developing infarct. We conclude that early, rather than delayed, expression of HSP-32 is a sensitive indicator of glial activation induced specifically by CGS-19755.     

Neuronal immunoreactivity for mannose-binding lectin after venous occlusion-induced focal cerebral ischemia in rats

A recent research reveals that complement activation exacerbates cerebral infarction. However, involvement of the lectin pathway, (the third complement activation pathway) in cerebral ischemia is not well studied. In this study, we investigated the appearance of mannose-binding lectin (MBL) in ischemic brain tissue. Male Wistar rats (n = 25) were divided into three groups: untreated control, sham, and vein occlusion (VO). Rats in the VO group had two adjacent photochemically occluded cortical veins. Regional cerebral blood flow (rCBF) was measured in the sham and VO groups. Rats were perfusion-fixed at 72 h in the sham group and at 3, 24, and 72 h after inducing ischemia in the VO group. Neuronal immunoreactivity for MBL, C1q, C3, and C5b-9 was graded on a scale from 0 (no staining) to 4 (strong immunoreactivity). rCBF significantly decreased in the VO group compared to the sham group. Immunohistochemical staining results were negative in the control group. MBL immunoreactivity was significant increased at 24 and 72 h after inducing ischemia in the VO group compared to the sham group. After inducing ischemia, C1 immunoreactivity was significantly increased at 3, 24, and 72 h, while C3 immunoreactivity increased at 72 h. C5b-9 immunoreactivity exhibited a tendency for only positive staining. In brain tissue after focal cerebral ischemia, MBL expression, as well as C1q and C3, appeared at 3 h, peaked at 24 h, and was maintained at 72 h. These results suggest that an MBL inhibitor administered during the relatively early ischemic phase might attenuate tissue damage.     

Serial magnetic resonance imaging in experimental primate stroke: Validation of MRI for pre-clinical cerebroprotective trials

Abstract

Precise assessment of stroke outcome is critical for pre-clinical testing of cerebroprotective strategies. Differences in stroke volume measured by various magnetic resonance imaging (MRI) techniques are documented in humans, but not well described in experimental primate stroke. This study characterizes changes in stroke volume using serial MRI scans in a baboon model of reperfused cerebral ischemia. The location/area of hyperintensity on MRI corresponded with the TTC-stained infarct region. T2-weighted fast spin echo (T2W FSE), fluid attenuated inversion recovery (FLAIR), and diffusion weighted imaging (DWI) showed a decrease in infarct volume between 72 h and nine days post-ischemia (p = ns, p = 0.029, and p = 0.006). T2W FSE and FLAIR demonstrated an increase in infarct volume from 24 h to nine days postischemia, while DWI displayed a decrease over the same period. While early T2W FSE, FLAIR, and DWI all correlated with late infarct volume (p < 0.001), 72 h T2W FSE was the best direct measure (2.39% ± 1.40% unity deviation). Serial MRI in a nonhuman primate model of focal cerebral ischemia recapitulates findings in clinical stroke. MRI at 72 h accurately predicts late infarct volume.     

Importance of Schedule of Administration in the Therapeutic Efficacy of Guanosine: Early Intervention After Injury Enhances Glutamate Uptake in Model of Hypoxia-ischemia

Perinatal cerebral hypoxia–ischemia (HI) is an important cause of mortality and neurological disabilities such as cerebral palsy, epilepsy, and mental retardation. The potential for neuroprotection in HI can be achieved mainly during the recovery period. In previous work, we demonstrated that guanosine (Guo) prevented the decrease of glutamate uptake by hippocampal slices of neonatal rats exposed to a hypoxic-ischemic (HI) insult in vivo when administrated before and after insult. In the present study, we compared the effect of Guo administration only after HI using various protocols. When compared with the control, a decrease of [³H] glutamate uptake was avoided only when three doses of Guo were administered immediately, 24 h and 48 h after insult, or at 3 h, 24 h, and 48 h after injury or at 6 h, 24 h, and 48 h after HI. These findings indicate that early Guo administration (until 6 h) after HI, in three doses may enhance glutamate uptake into brain slices after hypoxia/ischemia, probably resulting in decreased excitotoxicity.     

Recombinant fusion protein linking factor VIIa with albumin (rVIIa­FP): Tissue distribution in rats

Introduction

A novel fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) is currently undergoing clinical investigations.

Objective

This study was conducted to examine the biodistribution of rVIIa-FP in comparison to recombinant factor VIIa (rFVIIa).

Materials and Methods

[³H]-rVIIa-FP (10 mg kg^− 1) or [³H]-rFVIIa (1.6 mg kg^− 1) were administered intravenously to rats, followed by quantitative whole-body and knee joint autoradiography for 24 ([³H]-rFVIIa) or 240 ([³H]-rVIIa-FP) hours post-dose. Pharmacokinetic and excretion balance analyses were performed.

Results

In contrast to [³H]-albumin, the tissue distributions of [³H]-rVIIa-FP and [³H]-rFVIIa were similar. Within the knee, both were rapidly present within synovial and mineralized regions. Importantly, rVIIa-FP- and albumin-derived radioactivity were detectable up to 72–120 hours, whereas [³H]-rFVIIa signals were already close to detection limits at 24 hours. The longest rVIIa-FP retention times were observed in bone marrow and endosteum, in which the retention times were up to 5 times longer for rVIIa-FP compared with rFVIIa. Up to 8 hours post-dose, 100% of radioactivity was assigned to unchanged [³H]-rVIIa-FP. Elimination of both proteins occurred primarily via the urine.

Conclusions

The data suggest that the FVIIa moiety is directing rVIIa-FP’s tissue distribution while the albumin moiety is responsible for the prolonged tissue retention. Importantly, rVIIa-FP is highly concentrated and retained over a long period in the growth plate of the knee joint − a vulnerable site in haemophilia patients. Overall, these improved tissue distribution characteristics of rVIIa-FP may enhance compliance and allow a more convenient dosing frequency.     

Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics

The present study focuses on the temporal calcium significance in middle cerebral artery occluded (2 h ischemia)-reperfused (70 h reperfusion) rats treated with nimodipine (NM) through concurrent measurements of excitotoxicity, bioenergetics and neurobehavioural paradigms. Further, the suitable therapeutic time window of calcium channel antagonism in stroke was also ascertained. NM (5 mg/kg, i.p.) was administered at pre (30 min before the induction of ischemia), during (1 h following occlusion of MCA) and post-ischemic (3 h after begin of reperfusion) states. The magnitude of neuroprotection in terms of excitotoxicity (glutamate, glutamine synthetase, Na⁺K⁺ATPase), bioenergetics (ATP, NAD⁺) and neurobehavioural paradigms (neurological score and open field exploratory behaviour) were measured and compared to ensure the therapeutic time-window of NM in stroke. Middle cerebral artery occlusion-reperfusion (MCAO/R) was found to elevate glutamate, glutamine synthetase levels and deplete Na⁺K⁺ATPase activity in the vehicle treated group (IR group). Significant decrease in bioenergetics such as ATP and NAD⁺ levels was also observed. Further, IR group demonstrated grievous oxidative stress (increase in lipid peroxidation, protein carbonyl content, nitrite/nitrate levels and decrease in superoxide dismutase and glutathione levels) along with anxiogenic behaviour, neurological deficits and neuronal damage and decreased nuclear to cytoplasm ratio in CA1 hippocampal region. Post-ischemic NM administration reversed the excitotoxicity, neurobehavioural and histopathological alterations significantly, but it restored bioenergetics level in MCAO/R rats only partially.These findings were further confirmed with the combination treatment (CT) of post-ischemic NM and pre-ischemic memantine (MN) administration, since MN showed protective effect in the pre-ischemic administration (Babu and Ramanathan, 2009). The failure of NM to forefend the neurodegeneration on pre- and during-ischemic administration suggests that the initial phase damages in ischemic-reperfusion (IR) might be mediated through other mechanism(s) such as glutamergic overstimulation or reverse operation of glutamate transporters. From the present study, it is concluded that calcium plays a crucial role in post-ischemic status and the suitable therapeutic time window of calcium antagonism is the post-ischemic state.     

Pituitary adenylate cyclase-activating polypeptide plays a key role in nitroglycerol-induced trigeminovascular activation in mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (PAC1, VPAC) are present in sensory neurons and vascular smooth muscle. PACAP infusion was found to trigger migraine-like headache in humans and we showed its central pro-nociceptive function in several mouse pain models. Nitroglycerol (NTG)-induced pathophysiological changes were investigated in this study in PACAP gene-deleted (PACAP^−/−) and wildtype (PACAP^+/+) mice. Chemical activation of the trigeminovascular system was induced by 10 mg/kg i.p. NTG. Light-aversive behavior was determined in a light-dark box, meningeal microcirculation by laser Doppler blood perfusion scanning and the early neuronal activation marker c-Fos with immunohistochemistry. NTG-induced photophobia both in the early (0-30 min) and late phases (90-120 min) due to direct vasodilation and trigeminal sensitization, respectively, was significantly reduced in PACAP^−/− mice. Meningeal blood flow increased by 30-35% during 4 h in PACAP^+/+ mice, but only a 5-10% elevation occurred from the second hour in PACAP^−/− ones. The number of c-Fos expressing cells referring to neuronal activation in the trigeminal ganglia and nucleus caudalis significantly increased 4 h after NTG in PACAP^+/+, but not in PACAP^−/− animals. Similar PAC1 receptor immunostaining was detected in both groups, which did not change 4 h after NTG treatment. PACAP-38 (300 μg/kg, i.p.) produced photophobia similarly to NTG and 30% meningeal vasodilatation for 30 min in PACAP^+/+, but not in PACAP^−/− mice. It significantly increased neural activation 4 h later in the trigeminal ganglia of both groups, but in the nucleus caudalis of only the PACAP^+/+ mice.We provide the first experimental results that PACAP is a pivotal mediator of trigeminovascular activation/sensitization and meningeal vasodilation related to migraine.     

Time course of IL-6 expression in experimental CNS ischemia

Abstract

lnterleukin-6 (IL-6) appears to be an important modulator of the inflammatory response associated with CNS ischemia. Clinically, IL-6 values obtained in the first week post-stroke have been shown to correlate with infarct size and outcome. In this study we used a focal reversible stroke model to investigate the time course and relationship to outcome of IL-6 production in plasma, brain and CSF. Reversible middle cerebral artery occlusion or sham surgery was produced in 50 adult Swiss Webster mice by advancing an 8-0 filament into the internal carotid artery for 2 h (sham 1 min). At 3, 6, 12, 24, and 72 h (8 each ischemia; 2 each sham) groups of animals were evaluated on a 28 point clinical scale, blood and CSF obtainedand the brains were evaluated for infarct volume and IL-6 mRNA levels. Serum levels of IL-6 (ELISA mean ± SD; undetectable in controls) overall sham group, 102 ±87; 3 h, 908 ±494*pg ml^–1; 6 h, 1079±468*pgml^–1; 12h, 980±221 * pg ml^–1; 24 h, 320± 314*pgml^–1; 72 h, 20±30*pg ml^–1 (*p < 0.05 to sham). CSF levels (ELISA) overall sham group, 10±18; 3 h, 379±210*pgml^–1; 6 h, 157±61*pg ml^–1; 12 h, 136± 88*pgml^–1; 24 h, 127± 99 pg ml^–1; 72 h, 72±9* pg ml^–1 (*p < 0.05 to sham). Brain IL-6 mRNA levels overall sham group, 20; 3 h, 480; 6 h, 599; 12 h, 7960; 24 h, 20267; 72 h, 0. There was an overall R² of 0.20 between plasma and CSF IL-6. There was an overall R² of 0.13 and 0.20 between infarct size and serum and CSF IL-6 level respectively, and an overall R2 of 0.10 and 0.17 between neurologic function and serum and CSF IL-6 level respectively. These findings confirm that IL-6 values increase following CNS ischemia with peak serum and CSF levels occurring before brain values. CSF IL-6 levels had a stronger correlation with neurologic function and infarct size than serum. [Neurol Res 1999; 21: 287-292]     

A meta-analysis of two genome-wide association studies identifies 3 new loci for alcohol dependence

Family, twin and adoption studies have clearly demonstrated that genetic factors are important in modulating the vulnerability to alcohol dependence. Several genome-wide association (GWA) studies of alcohol dependence have been conducted; however, few loci have been replicated. A meta-analysis was performed on two GWA studies of 1283 cases of alcohol dependence and 1416 controls in Caucasian populations. Through meta-analysis we identified 131 SNPs associated with alcohol dependence with p < 10⁻⁴. The best novel signal was rs6701037 (p = 1.86 × 10⁻⁷) at 1q24-q25 within KIAA0040 gene while the second best novel hit was rs1869324 (p = 4.71 × 10⁻⁷) at 2q22.1 within THSD7B. The third novel locus was NRD1 at 1p32.2 (the top SNP was rs2842576 with p = 7.90 × 10⁻⁶). We confirmed the association of PKNOX2 at 11q24.4 with alcohol dependence. The top hit of PKNOX2 (rs750338 with p = 1.47 × 10⁻⁶) in the meta-analysis was replicated with the Australian Twin-Family Study of 778 families (p = 1.39 × 10⁻²) Furthermore, several flanking SNPs of the top hits in the meta-analysis demonstrated borderline associations with alcohol dependence in the family sample (top SNPs were rs2269655, rs856613, and rs10496768 with p = 4.58 × 10⁻³, 2.1 × 10⁻⁴, and 2.86 × 10⁻³ for KIAA0040, NRD1 and THSD7B, respectively). In addition, ALK, CASC4, and SEMA5A were strongly associated with alcohol dependence (p < 2 × 10⁻⁵) in the meta-analysis. In conclusion, we identified three new loci (KIAA0040, THSD7B and NRD1) and confirmed the previous association of PKNOX2 with alcohol dependence. These findings offer the potential for new insights into the pathogenesis of alcohol dependence.     

LAU-0901, a novel platelet-activating factor antagonist, is highly neuroprotective in cerebral ischemia

Platelet-activating factor (PAF) is a bioactive phospholipid that accumulates during ischemia-reperfusion and is involved in the activation of platelets, neutrophils, and pro-inflammatory signaling. PAF has been suggested to enhance brain ischemia-reperfusion damage. LAU-0901, a novel PAF receptor antagonist, was examined in models of focal cerebral ischemia in rats and mice. Sprague–Dawley rats were anesthetized and received 2-hour middle cerebral artery occlusion (MCAo) by intraluminal suture. LAU-0901 (30, 60, 90 mg/kg; n = 9–11) or vehicle (n = 11) was administered i.p. at 2 h after onset of MCAo. The neurological status was evaluated at 60 min, and on days 1, 2, 3 and 7 after MCAo. In the dose–response study in mice, C57BL/6 mice were anesthetized and received 1 h MCAo by intraluminal suture. LAU-0901 (15, 30, 60 mg/kg; n = 7–9) or vehicle (n = 8) was given i.p. at 1 h after onset of MCAo. Local cerebral blood flow (LCBF) was measured at 1, 2, 4, and 6 h after MCAo in mice. LAU-0901 treated rats showed improved neurological score throughout the 7-day survival period. LAU-0901 treatment (30, 60 and 90 mg/kg) reduced total corrected infarct volume compared to vehicle rats by 76, 88 and 90%, respectively. Mice treated with LAU-0901 (30 and 60 mg/kg) reduced total infarction by 29% and 66%, respectively. LCBF was improved by treatment with LAU-0901 (30 mg/kg) by 77% of baseline at 6 h. In conclusion, we demonstrate for the first time that LAU-0901 improves behavioral scores, LCBF and reduces infarct volume after focal cerebral ischemia in rats and mice. Thus, this PAF receptor antagonist exhibits potent and sustained neuroprotection that may be of value for the design of stroke therapies.     

Viability thresholds and the penumbra of focal ischemia

The classic concept of the viability thresholds of ischemia differentiates between two critical flow rates, the threshold of electrical failure and the threshold of membrane failure. These thresholds mark the upper and lower flow limits of the ischemic penumbra which is thought ot suffer only functional but not structural injury. Recent studies of the functional and metabolic disturbances suggest a more complex pattern of thresholds. At declining flow rates, protein synthesis is inhibited at first (at a threshold of about 0.55 ml/gm/min), followed by a stimulation of anaerobic glycolysis (at 0.35 ml/gm/min), the release of neurotransmitters and the beginning disturbance of energy metabolism (at about 0.20 ml/min), and finally the anoxic depolariztion (<0.15 ml/gm/min). The penumbra, as defined by the classic flow thresholds, does not remain viable for extended periods. Since viability of the tissue requires maintenance of energy-dependent metabolic processes, penumbra is redefined as a region of constrained blood supply in which the energy metabolism is preserved. Imaging of the penumbra by combining autoradiographic cerebral blood flow measurements with bioluminescent images of adenosine triphosphate (ATP) demonstrates a gradual expansion of the infarct core (in which ATP is depleted) into the penumbra until, after a few hours, the penumbra has disappeared. It is suggested that the limited survival of the penumbra is due to periinfarct depolarizations, which result in repeated episodes of tissue hypoxia, because the increased metabolic workload is not coupled to an adequate increase of collateral blood supply. This explains pharmacological suppression of periinfarct depolarizations lowering the threshold of metabolic disturbances and reducing the volume of the ischemic infarct.     

Characterization of mitochondrial glutaminase and amino acids at prolonged times after experimental focal cerebral ischemia

The mitochondrial enzyme glutaminase is a significant contributor to extracellular glutamate after neuronal injury in vitro [R. Newcomb, X. Sun, L. Taylor, N. Curthoys, R.G. Giffard, Increased production of extracellular glutamate by the mitochondrial glutaminase following neuronal death, J. Biol. Chem. 272 (1997) 11276–11282.]. As a step towards characterizing the role of the enzyme in neuronal injury in vivo, glutaminase activity was measured in central and peripheral regions of the ischemic distribution in rat brain at 6, 24, and 48 h after permanent focal ischemia. Although glutaminase activity decreases in the central ischemic area, significant activity remains in peripheral areas of evolving damage, even after 24 and 48 h ischemia. Western blots show no detectable change in glutaminase molecular weight or total immunoreactivity, regardless of the degree of inactivation. Significant amounts of glutamine remain in ischemic tissue at prolonged times after focal ischemia, while reductions in tissue amounts of glutamate are highly correlated with decreases in glutaminase activity. In vivo microdialysis probes were inserted into the ischemic periphery after 24 h focal ischemia. Glutamate is significantly elevated in these dialysates. Perfusion of the glutaminase substrate glutamine and the enzyme activator phosphate results in further and specific elevations in dialysate glutamate. In sum, significant mitochondrial glutaminase activity remains in the periphery of the ischemic lesion at 24 and 48 h, where it can contribute directly to elevated extracellular glutamate. Inactivation of the glutaminase in central areas of the ischemic lesion does not involve significant proteolytic degradation, and likely involves a specific molecular event.     

Total oligomeric flavonoids of Cyperus rotundus ameliorates neurological deficits, excitotoxicity and behavioral alterations induced by cerebral ischemic-reperfusion injury in rats

Interactions between neurons and astrocytes play a critical role in the central nervous system homeostasis. Cyperus rotundus (family: Cyperaceae), a traditional Indian medicinal herb, used as nervine tonic and nootropic in the Ayurvedic system of medicine. The present study was undertaken to investigate the neuroprotective effect of total oligomeric flavonoids (TOFs), prepared from C. rotundus, in rat model of cerebral ischemia and reperfusion. Male Sprague Dawley rats (290-340 g) were subjected to middle cerebral artery occlusion (MCAO) for 2 h and reperfusion for 70 h. Experimental animals were divided into four groups: Group I - sham operated (n = 7); Group II - vehicle treated ischemic-reperfusion (IR) (n = 9), and Group III and IV - TOFs treated (100 and 200 mg/kg body weight, p.o., respectively; n = 7 in each group). Vehicle or TOFs were pretreated for four days before the induction of ischemia and continued for next three days after the ischemia i.e. treatment was scheduled totally for a period of 7 days. MCAO surgery was performed on day 4, 1 h after TOFs administration. Neuroprotective effect of TOFs was substantiated in terms of neurological deficits, excitotoxicity (glutamate, glutamine synthetase and Na⁺K⁺ATPase levels), oxidative stress (malondialdehyde, super oxide dismutase, and glutathione) and neurobehavioral functions in the experimental animals. TOFs decreased glutamate, glutamine synthetase (GS) and increased Na⁺K⁺ATPase activity in a dose dependent manner when compared to the IR rats. Treatment with TOFs significantly reduced the neurological deficits and reversed the anxiogenic behavior in rats. Further, it also significantly decreased MDA and increased superoxide dismutase (SOD) and glutathione content in brains of experimental rats. Histopathological examination using cresyl violet staining revealed the attenuation of neuronal loss by TOFs in stroke rats. The present study demonstrates the unswerving involvement of TOFs on ischemia-reperfusion triggered biochemical alterations in MCAO/R rats. Hence, TOFs might be an attractive candidate for further studies in the development of new drugs for cerebral stroke treatment.     

Asphyxia induced by umbilical cord occlusion alters glutamatergic and GABAergic synaptic transmission in neurons of the superior colliculus in fetal rats

Using optical recordings, we studied the effects of asphyxia on intracellular Cl⁻ and Ca²⁺ concentrations ([Cl⁻]i; [Ca²⁺]i) in the superior colliculus of fetal rats, which were connected via the umbilical cord to the dam. Acute asphyxia was induced by umbilical cord occlusion. The number of fetal superior colliculus neurons showing GABA-mediated increases in [Cl⁻]i (leading to hyperpolarization) following local synaptic electrical stimulation had decreased by 3 h post-asphyxiation, while the number showing GABA-mediated decreases in [Cl⁻]i (leading to depolarization) increased. [Ca²⁺]i rise, which occurred after acute asphyxiation, was antagonized by both non-NMDA and NMDA receptor antagonists. The increase in [Ca²⁺]i following focal superior colliculus stimulation was markedly attenuated at 3 h post-asphyxiation.