Proteasome inhibitor PS519 reduces infarction and attenuates leukocyte infiltration in a rat model of focal cerebral ischemia

BACKGROUND AND PURPOSE: Reperfusion brain injury after cerebral ischemia is associated with a developing inflammatory response at the site of infarction. Proteasome inhibitors block nuclear factor-kappaB activation and provide anti-inflammatory effects in several animal models of peripheral inflammation. We tested the novel proteasome inhibitor PS519 in a rat model of transient focal ischemia to establish its pharmacodynamics as a neuroprotection treatment and related effects on leukocyte infiltration. METHODS: Rats were subjected to 2 hours of focal cerebral ischemia by means of the filament method of middle cerebral artery occlusion (MCAo). After either 22 or 70 hours of reperfusion, infarct size was measured and neurological function, electroencephalographic (EEG) activity, and/or neutrophil and macrophage infiltration was quantified. PS519 was administered in a single intravenous bolus at 2 hours after MCAo. In addition, the therapeutic window for PS519 was estimated by delaying treatment for 4 or 6 hours after MCAo. RESULTS: Dose-response analysis of infarct volume at 24 hours revealed that PS519 neuroprotection approached 60%, and clinical evaluations showed significant improvements in neurological function and EEG activity. Neutrophil infiltration at 24 hours was also significantly decreased in cortical and striatal infarcted tissue of PS519-treated rats. Delaying the PS519 treatment up to 4 hours continued to result in significant neuroprotection. In the 72-hour injury model, infarction was reduced 40% by PS519, and significant improvements in neurological function and EEG recovery were again measured. Considerable reductions in both neutrophil and macrophage infiltration were evident. CONCLUSIONS: PS519 mitigates infarction and improves neurological recovery in brain-injured rats, an effect in part caused by a reduction in the leukocyte inflammatory response.     

Delayed treatment with nicotinamide (Vitamin B(3)) improves neurological outcome and reduces infarct volume after transient focal cerebral ischemia in Wistar rats

BACKGROUND AND PURPOSE: We have previously shown that nicotinamide (NAm) acutely reduces brain infarction induced by permanent middle cerebral artery occlusion (MCAo) in rats. In this study, we investigate whether NAm may protect against ischemia/reperfusion injury by improving sensory and motor behavior as well as brain infarction volumes in a model of transient focal cerebral ischemia. METHODS: Forty-eight male Wistar rats were used, and transient focal cerebral ischemia was induced by MCAo for 2 hours, followed by reperfusion for either 3 or 7 days. Animals were treated with either intraperitoneal saline or NAm (500 mg/kg) 2 hours after the onset of MCAo (ie, on reperfusion). Sensory and motor behavior scores and body weight were obtained daily, and brain infarction volumes were measured on euthanasia. RESULTS: Relative to treatment with saline, treatment with NAm (500 mg/kg IP) 2 hours after the onset of transient focal cerebral ischemia in Wistar rats significantly improved sensory (38%, P<0.005) and motor (42%, P<0.05) neurological behavior and weight gain (7%, P<0.05) up to 7 days after MCAo. The cerebral infarct volumes were also reduced 46% (P<0.05) at 3 days and 35% (P=0.09) at 7 days after MCAo. CONCLUSIONS: NAm is a robust neuroprotective agent against ischemia/reperfusion-induced brain injury in rats, even when administered up to 2 hours after the onset of stroke. Delayed NAm treatment improved both anatomic and functional indices of brain damage. Further studies are needed to clarify whether multiple doses of NAm will improve the extent and duration of this neuroprotective effect and to determine the mechanism(s) of action underlying the neuroprotection observed. Because NAm is already used clinically in large doses and has few side effects, these results are encouraging for the further examination of the possible use of NAm as a therapeutic neuroprotective agent in the clinical treatment of acute ischemic stroke.     

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.     

Persistent neuroprotection with prolonged postischemic hypothermia in adult rats subjected to transient middle cerebral artery occlusion

Postischemic hypothermia provides long-lasting neuroprotection against global cerebral ischemia in adult rats and gerbils. Studies indicate that hypothermia must be prolonged (e.g., 24 h) to indefatigably salvage hippocampal CA1 neurons. Delayed hypothermia also reduces focal ischemic injury. However, no study has examined long-term outcome following postischemic hypothermia in adult animals. Furthermore, most studies examined only brief hypothermia (e.g., 3 h). Since previous studies may have overestimated long-term benefit and have likely used suboptimal durations of hypothermia, we examined whether prolonged cooling would attenuate infarction at a 2-month survival time following middle cerebral artery occlusion (MCAo) in rats. Adult male Wistar rats were implanted with telemetry brain temperature probes and later subjected to 30 min of normothermic MCAo (contralateral to side of probe placement) or sham operation. Ischemia was produced by the insertion of an intraluminal suture combined with systemic hypotension (60 mm Hg). Sham rats and one ischemic group controlled their own postischemic temperature while another ischemic group was cooled to 34 degrees C for 48 h starting at 30 min following the onset of reperfusion. The infarct area was quantified after a 2-month survival time. Normothermic MCAo resulted in almost complete striatal destruction (91% loss +/- 12 SD) with extensive cortical damage (36% +/- 16 SD). Delayed hypothermia treatment significantly reduced cortical injury to 10% +/- 10 SD (P < 0.001) while striatal injury was marginally reduced to 79% loss +/- 17 SD (P < 0.05). Delayed hypothermia of only 34 degrees C provided long-lasting cortical and striatal protection in adult rats subjected to a severe MCAo insult. These results strongly support the clinical assessment of hypothermia in acute stroke.     

Neuroprotection by tamoxifen in focal cerebral ischemia is not mediated by an agonist action at estrogen receptors but is associated with antioxidant activity

We have previously shown that tamoxifen can induce marked neuroprotection after middle cerebral artery occlusion (MCAo) in rats and have described two possible mechanisms of action: namely, inhibition of EAA release and inhibition of nNOS activity. In this study we tested other potential mechanisms. Namely, agonist action at estrogen receptors and an antioxidative action. Tamoxifen-treated rats had significantly improved neurobehavioral deficit scores after 24 h and showed approximately 75% reduced infarct volumes. These were unaffected by ICI 182,780 (a high affinity and pure receptor antagonist) administered intravenously, or intracisternally to avoid possible lack of brain penetration, 15 min before intravenous administration of tamoxifen. In rats subjected to 2 h MCAo followed by 22 h reperfusion, 1.8-fold and 2.9-fold increases of F(2)-IsoPs and F(4) neuroprostanes, respectively, as relatively stable markers of oxidative damage, were measured in the ischemic hemisphere compared with the corresponding contralateral hemisphere or sham controls. Tamoxifen given at 3 h after the start of ischemia reduced the IsoPs and NeuroPs to sham control levels, and also inhibited their production by chemically induced lipid peroxidation in brain homogenates. These data are consistent with at least part of tamoxifen's marked neuroprotection in focal cerebral ischemic injury being due to its antioxidant activity but not by an acute action on estrogen receptors (212 words).     

Neuroprotective effects of the sodium channel blocker RS100642 and attenuation of ischemia-induced brain seizures in the rat

Seizurogenic activity develops in many patients following brain injury and may be involved in the pathophysiological effects of brain trauma and stroke. We have evaluated the effects of the use-dependent sodium channel blocker RS100642, an analog of mexiletine, as a neuroprotectant and anti-seizure agent in a rat model of transient middle cerebral artery occlusion (MCAo). Post-injury treatment with RS100642 (0.01-5.0 mg/kg) dose-dependently reduced brain infarction, improved functional recovery of electroencephalographic (EEG) power, and improved neurological outcome following 2 h of MCAo and 24 h recovery. This effect was more potent and offered a larger reduction of brain infarct volume than a maximal neuroprotective dose of mexiletine (10.0 mg/kg). Furthermore, brain seizure activity recorded following 1 h MCAo and 72 h of recovery in injured rats was either completely blocked (30 min pre-MCAo treatment) or significantly reduced (30 min post-MCAo treatment) with RS100642 (1.0 mg/kg) treatment resulting in greater than 60% reduction of core brain infarct. These results indicate that brain seizure activity during MCAo likely contributes to the pathophysiology of brain injury and that RS100642 may be an effective neuroprotective treatment not only to decrease brain injury but also to reduce the pathological EEG associated with focal ischemia.     

Protective Effect of Zonisamide, an Antiepileptic Drug, Against Transient Focal Cerebral Ischemia with Middle Cerebral Artery Occlusion-Reperfusion in Rats

Summary: Purpose: The antiepileptic effects of zonisamide (ZNS) have been well documented experimentally and clinically. The purpose of this study was to examine whether ZNS reduces cerebral damage after transient focal ischemia in rats.
Methods : Ischemia was induced by a transient occlusion of the left middle cerebral artery (MCA) with a 3-0 nylon monofilament for 90 min. Neurological evaluation was performed by measuring the event of neurological deficit of the contralateral forepaw and hindpaw at 10 min and 1 day after MCA occlusion (MCAo). Brain infarct size was determined by measuring triphenyltetrazonium chloridenegative stained area of the serial brain sections 1 day after MCAo.
Results : The pre- or postischemic treatment with ZNS [(10–100 mgkg P.o.), 30 min before and 4 h after or 15 min and 4 h after the occlusion] markedly reduced cerebral damage in the ipsilateral hemisphere and the neurological deficit induced by transient ischemia. The reducing effect on the damage was observed in the cortical and subcortical regions. Preischemic treatment with carbamazepine (CBZ 60 mgkg p.0. twice 30 min before and 4 h after MCAo) tended to reduce the cerebral damage and neurological deficit, but the lower dose (20 mg/kg p.o. twice) did not. Valproate (VPA 1,000 mgkg p.o. twice) also had no effect.
Conclusions : ZNS at the anticonvulsant dose, unlike CBZ and VPA, ameliorated the brain infarction and the event of neurological deficit after transient focal cerebral ischemia. These data suggest that ZNS has therapeutic potential in protecting against ischemic cerebral damage, such as stroke.     

Neuroprotective effect of 4-amino-1,8-napthalimide, a poly(ADP ribose) polymerase inhibitor in middle cerebral artery occlusion-induced focal cerebral ischemia in rat

In the present study, neuroprotective effect of 4-amino-1,8-napthalimide (4-ANI), a poly(ADP-ribose) polymerase (PARP) inhibitor was investigated in middle cerebral artery occlusion (MCAo)-induced focal ischemia. Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. After 22 h of reperfusion rats were evaluated for cerebral infarction, neurological deficits, brain NAD levels, and in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). Focal ischemia produced significant infarct volume (201 +/- 14 mm3), neurological scores (2 +/- 0.5) and 28 +/- 4.5% brain NAD depletion. Ischemia was associated with increased in TUNEL positive cells in brain sections indicating DNA fragmentation. 4-ANI treatment (1 and 3 mg/kg, i.p.) significantly decreased infarct volume to 35 +/- 7% and 70 +/- 6%, respectively. Neurological functions were also significantly improved at these doses. 4-ANI (3 mg/kg) completely reversed brain NAD depletion and significantly reduced the increase in the number of TUNEL positive cells. Nevertheless, 4-ANI treatment did not alter cerebral blood flow and blood pressure. Our study suggests 4-ANI is a potent neuroprotective agent in focal cerebral ischemia and its neuroprotective effects may be attributed to reduction of NAD depletion and DNA fragmentation.     

The selective A2A receptor antagonist SCH 58261 reduces striatal transmitter outflow,turning behavior and ischemic brain damage induced by permanent focal ischemia in the rat

Adenosine A(2A) receptor antagonists have been proved protective in different ischemia models. In this study we verified if the protective effect of the selective A(2A) antagonist, SCH 58261, could be attributed to the reduction of the excitatory amino acid outflow induced by cerebral focal ischemia. A vertical microdialysis probe was inserted into the striatum of male Wistar rats and, after 24 h, permanent right intraluminal middle cerebral artery occlusion (MCAo) was induced. Soon after waking, rats showed a definite contralateral turning behavior, which persisted up to 7 h after MCAo. During 4 h after MCAo, glutamate, aspartate, GABA, adenosine and taurine outflow increased. SCH 58261 (0.01 mg/kg, i.p.), administered 5 min after MCAo, suppressed turning behavior and significantly reduced the outflow of glutamate, aspartate, GABA and adenosine. At 24 h after MCAo, the rats showed severe sensorimotor deficit and damage in both the striatum and cortex. SCH 58261 significantly reduced cortical damage but did not protect against the sensorimotor deficit. The protective effect of SCH 58261 against turning behavior and increased outflow of excitatory amino acids in the first hours after MCAo suggests the potential utility of selective adenosine A(2A) antagonists when administered in the first hours after ischemia. Furthermore, this study, for the first time, proposes that turning behavior after permanent intraluminal MCAo, be used as a precocious index of neurological deficit and neuronal damage.     

Middle cerebral artery occlusion in the mouse by intraluminal suture coated with poly-L-lysine: neurological and histological validation.

The present study was conducted to validate a modified method of temporary focal cerebral ischemia in the mouse; neurobehavioral function and histopathological infarction were quantitated following various periods of middle cerebral artery occlusion (MCAo). Male C57BL/6 mice were anesthetized with 3% halothane in a mixture of 30%O2/70%N2O delivered by face mask and were subjected to 30- to 180-min of temporary middle cerebral artery occlusion (MCAo) by an intraluminal suture coated with poly-l-lysine. Twenty-eight of 40 mice showed an initial high-grade neurological deficit (30-min MCAo, n=7; 60-min, n=8; 120-min, n=8; 180-min, n=5) when examined during MCAo; these were used for subsequent study. One day after MCAo, behavioral function was re-evaluated, and brains were perfusion-fixed and infarct volumes were measured. The initial neurological deficit improved at 24 h in mice with 30- or 60-min of prior MCAo but tended to persist in mice with 120- or 180-min insults. Following each duration of ischemia, mice exhibited ipsilateral infarcts. Small, inconsistent predominantly subcortical infarcts were present after 30-min MCAo, while longer occlusion periods gave rise to consistent foci of subcortical infarction involving striatum, septum, thalamus, and hippocampus, as well as areas of frontoparietal cortical infarction. The major advantages of the improved intraluminal MCAo model reported here, incorporating sutures coated with poly-l-lysine, include: a 100% incidence of infarction of predictable location and size in mice having an initial neurological deficit. Periods of 60- to 180-min MCA occlusion in this model yield sufficiently reproducible sequelae to permit the effects of various therapeutic agents on neurological outcome and size of infarction to be readily studied.     

Citalopram attenuated neurobehavioral,biochemical, and metabolic alterations in transient middle cerebral artery occlusion model of stroke in male Wistar rats

Oxidative stress and inflammation are implicated as cardinal mechanisms of neuronal death following stroke. In the present study citalopram (Cit) was investigated in a 2 h middle cerebral artery occlusion (MCAo) model of stroke in male Wistar rats. Pretreatment, posttreatment (Post Cit) and pre plus posttreatment (Pre + Post Cit) with Cit were evaluated for its neuroprotective effect. In pretreatment protocol, effect of Cit at three doses (2, 4, and 8 mg/kg) administered i.p., 1 h prior to MCAo was evaluated using neurological deficit score (NDS), motor deficit paradigms, and cerebral infarction 24 h post‐MCAo. In posttreatment and pre plus posttreatment protocol, the effective dose of Cit (4 mg/kg) was administered i.p., 0.5 h post‐reperfusion (Post Cit) only, and 1 h prior to MCAo and again at 0.5 h post‐reperfusion (Pre + Post Cit), respectively. These two groups were assessed for NDS and cerebral infarction. Though NDS was significantly reduced in both Post Cit and Pre + Post Cit groups, significant reduction in cerebral infarction was evident only in Pre + Post Cit group. Infarct volume assessed by magnetic resonance imaging was significantly attenuated in Pre + Post Cit group (10.6 ± 1.1%) compared to MCAo control group (18.5 ± 3.0%). Further, Pre + Post Cit treatment significantly altered 17 metabolites along with attenuation of malondialdehyde, reduced glutathione, matrix metalloproteinases, and apoptotic markers as compared to MCAo control. These results support the neuroprotective effect of Cit, mediated through amelioration of oxidative stress, inflammation, apoptosis, and altered metabolic profile.     

A selective endothelin ET(A) receptor antagonist, SB 234551, improves cerebral perfusion following permanent focal cerebral ischemia in rats

In recent experimental studies, a selective antagonist of endothelin ET(A) receptors, SB 234551, improved neurological and histological outcome in both head trauma and transient focal cerebral ischemia. The present study was conducted to ascertain the degree to which hemodynamic alterations are responsible for this therapeutic effect in a model of permanent middle cerebral artery occlusion (MCAo) in rats. Anesthetized Sprague-Dawley rats were subjected to permanent MCAo by insertion of an intraluminal nylon suture coated with poly-L-lysine. The agent (SB 234551, 30 microg/kg/min = 1.8 mg/kg/h) or vehicle (PBS; 0.6 ml/h) was administered by i.v. infusion beginning 15 min after onset of MCAo and lasting for 23.75 h. Autoradiographic measurement of local cerebral blood flow (lCBF) was performed at 24 h. Physiological data were similar among groups. SB 234551 augmented perfusion by 1.7- to 1.8-fold in both the ischemic hemisphere and in the contralateral (non-ischemic) hemisphere when compared to vehicle-treated ischemic animals. In the ischemic hemisphere, the brain regions significantly benefited were those lying outside the zone of most dense ischemia (i.e., paramedian cortex and thalamus), while in the non-ischemic hemisphere all regions measured showed significant lCBF augmentation. This study demonstrates that SB 234551 therapy results in significant improvement of local cerebral perfusion in the ischemic as well as in the non-ischemic hemispheres after permanent MCAo.     

Effects of leukemia inhibitory factor on endogenous neural stem cell proliferation and glycoprotein-130 expression in a mouse model of cerebral infarction

Leukemia inhibitory factor (LIF) has been shown to promote proliferation of endogenous neural stem cells. In this study, we treated mice with cerebral infarction using LIF to investigate whether the LIF receptor subunit glycoprotein (gp)130 is involved in neuroprotection. After LIF treatment, the motor function of model mice was significantly improved. Immunofluorescence histochemistry showed increased numbers of endogenous neural stem cells surrounding the infarct foci. Western blot analysis revealed that gp130 expression was significantly decreased surrounding the infarcted foci. Results demonstrated that LIF promoted the proliferation of endogenous neural stem cells by inhibiting gp130 protein expression.     

缺血再灌注大鼠海马的神经干细胞移植：磷脂酰肌醇3/Akt通路激活和脑源性神经营养因子表达增加

背景：PI3K/Akt通路和BDNF在脑缺血动物模型和患者中表达异常,也成为的脑缺血的治疗靶点。神经干细胞在修复的潜在用途包括移植修复丢失的细胞和激活内源性细胞提供“自我修复。” 目的：观察神经干细胞植入对PI3K/Akt和BDNF在海马表达的影响,阐明神经干细胞植入对脑缺血的神经保护机制。 设计：完全随机分组,对照动物实验。 时间及地点：实验于2007-03-2008.09在南方医科大学基因工程研究所完成。 材料：E17的怀孕大鼠和雌性大鼠购自南方医科大学实验动物中心,兔抗PI3K的试剂盒购自北京博奥森生物科技公司,磷酸化Akt（Ser473）由美国Cell Signaling Technology提供,其他试剂由美国sigma和Santa Cruz公司提供。 方法：体外分离、培养大鼠NSCs,进行WTS-8、 BrdU检测。局灶性脑缺血模型,大脑中动脉线栓方法制作大鼠脑缺血再灌注模型。参照Longa氏5分评分方法,得分超过2分大鼠入选实验。2,3,5,氯化三苯基四唑（TTC）染色检测梗死体积。两天后大脑中动脉阻塞大鼠给与50000 E17的立体定向神经干细胞移植或5μgWortmannin至缺血海马旁。使用免疫印迹分析Akt（Ser473）,PI3K和BNDF的表达。 主要观察指标：脑组织神经干细胞移植激活PI3K/Akt通路参与促进神经组织的结构和功能恢复且上调脑源性神经营养因子的功能。 结果：神经干细胞治疗组脑梗死体积显着低于缺血模型组(P<0.01)、Wortmannin干预组(P<0.01)和神经干细胞+Wortmannin干预组(P<0.05)。在神经干细胞治疗组PI3K蛋白的水平比较显着高于脑缺血模型性(P<0.01)。在Akt的蛋白质水平（Ser473）和神经干细胞治疗组BNDF更为显著高于脑缺血模型性(P<0.01)。在治疗组的BNDF蛋白水平比较显着高于Wortmannin干预(P<0.01)神经干细胞+Wortmannin干预组(P<0.05)。 结论：在脑缺血过程中神经干细胞移植激活PI3K/Akt通路参与脑源性神经营养因子的基因表达。     

MRI of combination treatment of embolic stroke in rat with rtPA and atorvastatin

To test the hypothesis that combination treatment of embolic stroke with rtPA and statins improves the efficacy of thrombolytic therapy in rats. Rats subjected to embolic MCA occlusion (MCAo) were randomized into control (n = 10) and treatment (n = 9) groups. Four hours after MCAo, a combination of rtPA and atorvastatin (treatment) or saline (control) was administered. MRI measurements were performed on all animals at 2 h, 24 h and 48 h after MCAo. The patency of cerebral microvessels was examined using fluorescent microscopy. MRI images showed complete blockage of the right MCA and a reduction of CBF in the territory supplied by the MCA 2 h after MCAo for all animals. By 48 h after stroke, MRI showed that the decreased lesion size, elevated CBF and increased incidence of recanalization were found in treated rats compared with the control rats. The combination treatment significantly increased microvascular patency (16.3 +/- 5.5% vs. 12.4 +/- 3.5%, of field-of-view) and reduced the infarct volume (23.1 +/- 9.6% vs. 38.8 +/- 13.3%, of hemisphere). These data demonstrate that the co-administration of rtPA and atorvastatin 4 h after ischemia is efficacious and is reflected by the MRI indices of recanalization of the MCA, reduction of secondary microvascular perfusion deficits and reduction of the ischemic lesion.     

Wide therapeutic time window for nimesulide neuroprotection in a model of transient focal cerebral ischemia in the rat

Results from several studies indicate that cyclooxygenase-2 (COX-2) is involved in ischemic brain injury. The purpose of this study was to evaluate the neuroprotective effects of the selective COX-2 inhibitor nimesulide on cerebral infarction and neurological deficits in a standardized model of transient focal cerebral ischemia in rats. Three doses of nimesulide (3, 6 and 12 mg/kg; i.p.) or vehicle were administered immediately after stroke and additional doses were given at 6, 12, 24, 36 and 48 h after ischemia. In other set of experiments, the effect of nimesulide was studied in a situation in which its first administration was delayed for 3-24 h after ischemia. Total, cortical and subcortical infarct volumes and functional outcome (assessed by neurological deficit score and rotarod performance) were determined 3 days after ischemia. The effect of nimesulide on prostaglandin E(2) (PGE(2)) levels in the injured brain was also investigated. Nimesulide dose-dependently reduced infarct volume and improved functional recovery when compared to vehicle. Of interest is the finding that neuroprotection conferred by nimesulide (reduction of infarct size and neurological deficits and improvement of rotarod performance) was also observed when treatment was delayed until 24 h after ischemia. Further, administration of nimesulide in a delayed treatment paradigm completely abolished PGE(2) accumulation in the postischemic brain, suggesting that COX-2 inhibition is a promising therapeutic strategy for cerebral ischemia to target the late-occurring inflammatory events which amplify initial damage.     

Neuroprotective effect of combination of poly (ADP-ribose) polymerase inhibitor and antioxidant in middle cerebral artery occlusion induced focal ischemia in rats

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Punjab, India We have investigated the neuroprotective potential of combination of poly (ADP-ribose) polymerase inhibitor (nicotinamide or 3-aminobenzamide) and antioxidant (melatonin) in middle cerebral artery occlusion (MCAo) induced focal ischemia in rats. MCAo of 2 h followed by 22 h reperfusion produced large volume of cerebral infarction (mean +/- SEM 211.38 +/- 8.35 mm3), volume of edema (60 +/- 2 mm3) and neurological deficits (4.45 +/- 0.25). Combination of nicotinamide (500 mg kg(-1), i.p.) and melatonin (10 mg kg(-1), i.p.) significantly decreased infarct volume to 48 +/- 2.58 mm3 as compared to their individual drug (nicotinamide 76 +/- 12.49mm3, melatonin 76.17 +/- 1.24 mm3). A significant improvement was observed in edema volume and neurological deficits with this combination. Combination of 3-aminobenzamide (20 mg kg(-1), i.p.) and melatonin (10 mg kg(-1), i.p.) also produced similar reduction in infarction, edema and neurological score. These results indicate that the combination of poly (ADP-ribose) polymerase inhibitor and antioxidant produce enhanced neuroprotection. Clinical availability and wide therapeutic margin of nicotinamide and melatonin make them a promising drug combination for clinical evaluation in stroke patients.     

Caspase inhibitors reduce neuronal injury after focal but not global cerebral ischemia in rats

BACKGROUND AND PURPOSE: Studies show that blocking the activation of caspases by the caspase inhibitors z-VAD.FMK and z-DEVD.FMK can reduce ischemic neuronal injury after cerebral ischemia. Because the severity of ischemia was mild in some studies, we tested the efficacy of these caspase inhibitors on moderately severe but transient forebrain and focal ischemic insults in the rat. METHODS: Various regimens of z-VAD, z-DEVD, and control DMSO were given to rats subjected to either 4-vessel occlusion ischemia (4-VO, 10-minute occlusion, 7-day survival) or distal middle cerebral artery occlusion (MCAo, 90-minute occlusion, 22.5-hour survival). In global ischemia, treatments were given immediately after ischemia (experiment 1) or as preischemic and postischemic treatments (experiment 2). Three focal ischemia experiments were done. Injection times were 60 minutes into ischemia (experiment 1) and 60 minutes into ischemia plus 30 and 120 minutes after ischemia (experiment 2). Experiment 3 was identical to experiment 2 except that a 30-minute preischemia treatment was instituted. Core normothermia was maintained in all experiments during ischemia. However, in the last focal and global experiments, core and brain temperatures, respectively, were also measured after ischemia with telemetry probes. Because hyperthermia accompanied z-DEVD treatment, an extra z-DEVD-treated group (MCAo) was included with temperature clamped at normothermia. RESULTS: Neither z-VAD nor z-DEVD significantly reduced CA1 injury after global ischemia. In focal ischemia, both drugs significantly reduced infarction, but only in the third experiment, and the prevention of hyperthermia that accompanied z-DEVD treatment did not alter this. CONCLUSIONS: These results suggest a detrimental role of caspases in moderately severe focal but not global cerebral ischemia.     

Effects of delayed intrathecal infusion of an NMDA receptor antagonist on ischemic injury and peri-infarct depolarizations

The potent NMDA receptor antagonist, Conantokin-G (CGX-1007), a snail peptide, has an 8-h therapeutic window in rat focal cerebral ischemia. We hypothesized that the mechanism of neuroprotection is the inhibition of 'secondary phase' peri-infarct depolarizations (PIDs), recently shown to recur 6--24 h post-reperfusion. Rats were implanted with intrathecal (i.t.) catheters for drug delivery and DC-compatible electrodes for continuous PID monitoring and subjected to transient (2 h) middle cerebral artery occlusion. Four groups were studied. In two groups (C(40)C and C(20)C), continuous infusion of CGX--1007 was administered over 8--24 h post-occlusion at 0.1 microg/h (0.04 nmol/h) following either a 40- or 20-nmol bolus dose at 8 h. Another group (C(40)S) received the 40-nmol bolus followed by saline infusion, and a control group received saline. Intrathecal drug treatment reduced infarct volumes relative to controls by 61%, 31%, and 10% in C(40)C, C(40)S, and C(20)C groups, respectively, but also induced dose-dependent paralysis and elevated mortality. All rats had PID rates similar to the control group prior to treatment, but following treatment secondary phase PIDs were reduced by 47--57% in each drug group compared to controls. Because several animals exhibited PID inhibition but no neuroprotection, there was no significant correlation between these endpoints across groups. However, drug-treated animals that did not exhibit secondary phase PIDs prior to treatment had significantly smaller infarcts and reduced subsequent PID activity than corresponding control rats. Results suggest that post-reperfusion PIDs play a substantial, though still undefined pathogenic role in delayed maturation of cerebral infarction and NMDA receptor-targeted neuroprotection.