Reversible Focal Cerebral Ischemia Research Articles

Neuroprotective Effects of a Novel Inhibitor of c-Jun N-Terminal Kinase in the Rat Model of Transient Focal Cerebral Ischemia.

A novel specific inhibitor of c-Jun N-terminal kinase, 11H-indeno[1,2-b]quinoxalin-11-one oxime sodium salt (IQ-1S), has a high affinity to JNK3 compared to JNK1/JNK2. The aim of this work was to study the mechanisms of neuroprotective activity of IQ-1S in the models of reversible focal cerebral ischemia (FCI) in Wistar rats. The animals were administered with an intraperitoneal injection of IQ-1S (5 and 25 mg/kg) or citicoline (500 mg/kg). Administration of IQ-1S exerted a pronounced dose-dependent neuroprotective effect, not inferior to the effects of citicoline. Administration of IQ-1S at doses of 5 and 25 mg/kg reduced the infarct size by 20% and 50%, respectively, 48 h after FCI, whereas administration of citicoline reduced the infarct size by 34%. The administration of IQ-1S was associated with a faster amelioration of neurological status. Control rats showed a 2.0-fold increase in phospho-c-Jun levels in the hippocampus compared to the corresponding values in sham-operated rats 4 h after FCI. Administration of IQ-1S at a dose of 25 mg/kg reduced JNK-dependent phosphorylation of c-Jun by 20%. Our findings suggest that IQ-1S inhibits JNK enzymatic activity in the hippocampus and protects against stroke injury when administered in the therapeutic and prophylactic regimen in the rat model of FCI.

The dichotomy of memantine treatment for ischemic stroke: dose-dependent protective and detrimental effects.

Excitotoxicity is a major contributor to cell death during the acute phase of ischemic stroke but aggressive pharmacological targeting of excitotoxicity has failed clinically. Here we investigated whether pretreatment with low doses of memantine, within the range currently used and well tolerated for the treatment of Alzheimer's disease, produce a protective effect in stroke. A coculture preparation exposed to modeled ischemia showed cell death associated with rapid glutamate rises and cytotoxic Ca2+ influx. Cell death was significantly enhanced in the presence of high memantine concentrations. However, low memantine concentrations significantly protected neurons and glia via excitotoxic cascade interruption. Mice were systemically administered a range of memantine doses (0.02, 0.2, 2, 10, and 20 mg/kg/day) starting 24 hours before 60 minutes reversible focal cerebral ischemia and continuing for a 48-hour recovery period. Low dose (0.2 mg/kg/day) memantine treatment significantly reduced lesion volume (by 30% to 50%) and improved behavioral outcomes in stroke lesions that had been separated into either small/striatal or large/striatocortical infarcts. However, higher doses of memantine (20 mg/kg/day) significantly increased injury. These results show that clinically established low doses of memantine should be considered for patients ‘at risk' of stroke, while higher doses are contraindicated.

Significance of Experimental Infarct Size as an Indicator of Therapeutic Efficacy in Humans

To the Editor: We would like to make a comment not in particular to the article by Shimamura et al1 but to the use of infarct size in animal experiments as a guide to human cerebrovascular research. Many human studies of treatments with neuroprotective agents for acute ischemic stroke, which had good experimental potential, had disappointing clinical results. This failure can be attributed to many factors.2 One of the main problems is that reliance on infarct size measurement alone in animals can be misleading as an indicator of therapeutic efficacy in humans.3 We know for example that nitric oxide synthase (NOS) inhibitors have provided significant neuroprotection in experimental animals subjected to focal ischemia.4,5 Tirilazad mesylate is a potent inhibitor of lipid peroxidation caused by …

Neuroprotective Effects of Virally Delivered HSPs in Experimental Stroke

Heat shock proteins (HSPs) are molecular chaperones with essential roles in modulating the proteolytic machinery and accelerating cell repair. Heat shock protein overexpression has been observed in vivo and in vitro under stresses including heat, nutrient deprivation and ischemia. Experiments in in vivo models of stroke indicate that transgenically overexpressed or virally delivered HSPs can enhance cell survival, but cannot always reduce lesion size. This study aims to assess the effects of virally delivered HSPs in a rat middle cerebral artery occlusion model of reversible focal cerebral ischemia using noninvasive magnetic resonance imaging. Attenuated herpes simplex virus carrying HSP27, HSP70, or a LacZ control was microinjected into the striatum 3 days before ischemia. Multislice T(2)-weighted images at 24 h after ischemia indicated that lesion volume was reduced by 44% in HSP27-treated animals compared with controls (P = 0.019). No significant differences were found between HSP70-treated and control animals (P = 0.88). Immunohistochemistry and Western blots revealed that HSP27 and HSP70 expression levels were equally high in injected hemispheres, but only the former had an effect on lesion size. This is the first evidence of the efficacy of gene therapy with any viral vector expressing HSP27 in an experimental model of stroke.

Gene therapy for stroke using viral delivery of heat schock proteins

Heat shock proteins (HSPs) are molecular chaperones with essential roles in cellular function such as modulation of the proteolytic machinery and acceleration of cell repair. This study uses MRI to assess the effects of pre-ischaemic viral delivery of HSPs on lesion size in a rat middle cerebral artery (MCA) occlusion model of reversible focal ischaemia. Perfusion maps of a 2-mm brain slice within the MCA territory were acquired to measure cerebral blood flow (CBF) and multislice T2-weighted scans were used to determine lesion volume 24 hours after ischaemia. Male rats were anaesthetised with 2% isoflurane in 100% O2. Viral suspensions (2.5 l of 2106 pfu) of herpes simplex virus carrying HSP27 (n=6), HSP70 (n=6), or LacZ (n=6) as a control, were stereotactically injected into rat striatum. Three days post-injection, rats were re-anaesthetised for middle cerebral artery occlusion (MCAO) by intraluminal insertion of a 290-m-suture past the MCA junction. After 30 minutes the suture was completely withdrawn to reperfuse the tissue and rats were allowed to recover for 24 hours before scanning. Coronal images were obtained approximately 0.5 mm from bregma on a 2.35 Tesla horizontal bore SMIS magnet with 4020 mm FOV, 2 mm slice thickness and 12864 pixels. T1 and CASL (continuous arterial spin labelling) EPI sequences were run for quantitative CBF mapping 3 and multislice T2-weighted SE images (1 mm slice thickness, 9 slices, TE=120 ms, TR=1500 ms) were acquired for lesion definition. All animals were imaged under general anaesthetic (halothane 2% in a 70:30 N2O:O2 mix) with physiological monitoring (ECG, rectal temperature). Three days later, the brains were extracted and immunohistochemistry and Western blots were carried out in order to verify expression levels of virally delivered HSPs in the brain. Total lesion volume was reduced by 44.8% (p=0.02) in HSP27 treated animals compared to controls whereas no significant differences were found between HSP70 treated and control animals (p=0.88). CASL maps indicated that there was no significant difference in relative CBF 24 hours after reperfusion in normal and ischaemic hemispheres between the groups that could account for differences in lesion size. Histological analysis of brain sections showed widespread staining for HSPs in basal ganglia and cortex. Western blots performed 72 hours after MCAO revealed that levels of HSP expression in HSP injected hemispheres were 4 times higher than in LacZ injected controls. In conclusion, we show that non–invasive MRI techniques can detect a significant reduction in lesion size after HSP27 gene delivery in a rat model of reversible focal cerebral ischaemia.

SLOW-MAG.

SLOW-MAG.

Editorial Comment—Hyperglycemia and Early Reperfusion Therapy

Clinical studies of acute ischemic stroke support the conclusions of most experimental studies of focal cerebral ischemia in suggesting that admission hyperglycemia is associated with a worse clinical outcome.1 This association is more consistent in nonlacunar strokes2 and in experimental models of reversible focal cerebral ischemia.3 The acute ischemic penumbra might be preferentially susceptible to injury in hyperglycemic ischemia. Indeed, by use of MR methods, hyperglycemia in acute ischemic stroke was shown to promote the evolution of hypoperfused tissue to infarction and to do so by increased brain lactate …

N‐acetyl cysteine protects against injury in animal model of transient focal cerebral ischemia

Ischemic cerebrovascular disease (stroke) is one of the leading causes of death and long‐term disability worldwide (or developing countries). The purpose of this study is to investigate the therapeutic potential of N‐acetyl cysteine (NAC), a precursor of glutathione, and a potent antioxidant, in tissue protection against transient focal cerebral ischemia model in rats. Our laboratory has already demonstrated the efficacy of NAC against induction of proinflammatory cytokines, iNOS, and production of NO in cultured cells. In this study, 250–300 g male Sprague–Dawley rats were subjected to 45 min of reversible focal cerebral ischemia through middle cerebral artery (MCA) occlusion. The protective effect of NAC is supported by reduced infarct volume as well as the suppression of TNF‐α and iNOS in the treated animals. Pretreatment with NAC reduced the infarct size by 49.6% as compared to the saline treated group along with reducing the neurological evaluation score by more than 50%. Our results suggest that preadministration of NAC attenuates focal cerebral ischemia by protecting cells against free radical damage.Acknowledgements: Supported by NIH grants NS‐40144, NS‐40810, NS‐22576, NS‐34741 and NS‐37766.

Dose-dependent neuroprotection with tiagabine in a focal cerebral ischemia model in rat.

Enhancement of the inhibitory activity of GABA may protect ischemic neurons. We evaluated the neuroprotective effect of tiagabine, a novel GABA agonist, in reversible focal cerebral ischemia rats subjected to 2h middle cerebral artery (MCA) occlusion. Tiagabine was given at 10, 20 and 40 mg/kg, i.p., 2 h after the onset of reperfusion. We found that post-ischemia treatment with tiagabine improved neurobehavioral outcome and reduced brain infarction volume in a dose-dependent manner. The data suggest that post-ischemic administration of tiagabine is neuroprotective in the focal cerebral ischemia model.

Attenuation of free radical generation during reversible focal cerebral ischemia with the nitric oxide inhibitor, l-NAME ( l- NG-nitro- l-arginine methyl ester)

The role of oxygen free radical generation during reversible focal cerebral ischemia and its relationship to nitric oxide mediated mechanisms were examined. In this study, a left frontal cortex microdialysis probe was placed into the previously defined ischemic penumbra region and perfused with a salicylate/CSF solution in the presence or absence of the nitric oxide synthase (NOS) inhibitor l-NAME. Rats were then subjected to transient left hemisphere focal cerebral ischemia. Dialysate was collected at baseline and during the ischemic/reperfusion phase, and the hydroxylation products of salicylate were measured by HPLC with electrochemical detection. A significant elevation of free radical adduct formation was observed in the penumbra region during ischemia/reperfusion. This elevation was significantly attenuated by l-NAME during the reperfusion phase. Elevation of free radical adduct formation within the penumbra region during cerebral ischemia/reperfusion may be mediated in part by NOS-dependent mechanisms.

Quantitative T*2 and T?2 maps during reversible focal cerebral ischemia in rats: Separation of blood oxygenation from nonsusceptibility-based contributions

Quantitative imaging contrast is evaluated which allows the selective measurement of the blood oxygenation state during cerebral ischemia within a multiparametric imaging study on rats. In a first step, the ambiguities arising in T*(2)-weighted images due to T*(2) heterogeneity are eliminated by calculating T*(2) maps. Then, 1/T'(2) maps are calculated according to 1/T'(2) = 1/T*(2) - 1/T(2) to eliminate nonsusceptibility-induced changes of 1/T*(2) after the induction of stroke. This is of particular importance in the presence of vasogenic edema. The changes Delta(1/T'(2)) after the onset of ischemia selectively quantify the variations of the deoxyhemoglobin content during the development of the infarct. The presented results are not available from conventionally recorded parameters of a stroke study and, together with perfusion-weighted images, form a powerful combination to analyze the oxygen consumption and the metabolism of the tissue. Magn Reson Med 42:1027-1032, 1999.

Quantitative T*(2) and T'(2) maps during reversible focal cerebral ischemia in rats: separation of blood oxygenation from nonsusceptibility-based contributions.

Quantitative imaging contrast is evaluated which allows the selective measurement of the blood oxygenation state during cerebral ischemia within a multiparametric imaging study on rats. In a first step, the ambiguities arising in T*(2)-weighted images due to T*(2) heterogeneity are eliminated by calculating T*(2) maps. Then, 1/T'(2) maps are calculated according to 1/T'(2) = 1/T*(2) - 1/T(2) to eliminate nonsusceptibility-induced changes of 1/T*(2) after the induction of stroke. This is of particular importance in the presence of vasogenic edema. The changes Delta(1/T'(2)) after the onset of ischemia selectively quantify the variations of the deoxyhemoglobin content during the development of the infarct. The presented results are not available from conventionally recorded parameters of a stroke study and, together with perfusion-weighted images, form a powerful combination to analyze the oxygen consumption and the metabolism of the tissue. Magn Reson Med 42:1027-1032, 1999.

Increased intracellular calpain detection in experimental focal cerebral ischemia.

Calpains are intracellular proteinases whose proteolytic activity is directed mainly against the cytoskeleton and regulatory proteins. We studied the presence of calpain by immunohistochemistry in a rat model of reversible focal cerebral ischemia (3 h) at various times of reperfusion. The numbers of calpain-positive cells on the ischemic side were compared with the non-ischemic side. In controls only 2 +/- 1% cells were positive, whereas the cortex of the ischemic vs the non-ischemic side showed 88 +/- 3% vs 13 +/- 4% calpain-positive cells (p < 0.001), and the basal ganglia 47 +/- 3% vs 13 +/- 4% (p < 0.01) after 3 h ischemia and 24 h reperfusion. This is the first demonstration of elevated intracellular levels of calpains in areas of cerebral ischemia. Longer reperfusion resulted in an increase in calpain positivity.

Neuroprotective effects of combination therapy with tirilazad and magnesium in rats subjected to reversible focal cerebral ischemia.

Cell death after cerebral ischemia is mediated by release of excitatory amino acids, calcium influx into cells, and generation of free radicals. We examined the hypothesis that concurrent administration of tirilazad, a well-known antioxidant, and magnesium, an antagonist of calcium and excitatory amino acids, would result in a synergistic neuroprotective effect. Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and assigned to one of four treatment arms (n = 10 in each): 1) vehicle, 2) tirilazad, 3) MgCl2, or 4) tirilazad and MgCl2. Cortical blood flow was recorded using laser Doppler flowmetry. Functional deficits were quantified by performing daily neurological examinations. Infarct volume was assessed after 7 days. There was no difference in cortical blood flow among groups. Animals that received tirilazad or MgCl2 monotherapy had significantly better neurological function compared with control animals only on postoperative Days 3 and 4, whereas animals treated with both drugs had significantly better neurological function than did control animals from postoperative Days 2 through 7. Magnesium reduced total infarct volume by 25% (nonsignificant), tirilazad by 48% (P<0.05), and combination therapy by 59% (P<0.05) compared with control data. Combined therapy with antagonists of excitatory amino acids and free radicals provides better neuroprotection from the effects of transient focal ischemia than does therapy with either antagonist alone. In contrast to many experimental agents, tirilazad and magnesium offer the advantage of being licensed for clinical use. This drug combination could be of great benefit when administered before temporary artery occlusion in patients undergoing cerebrovascular surgery.

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Neuroprotective Effects of Combination Therapy with Tirilazad and Magnesium in Rats Subjected to Reversible Focal Cerebral Ischemia

Tizanidine is an effective agent in the prevention of focal cerebral ischemia in rats: an experimental study

BACKGROUND Focal cerebral ischemia secondary to cerebral vessel occlusion is still an important cause of mortality and morbidity. Excitatory neurotransmitters are gathered in the extracellular space during ischemia and initiate or stimulate a series of pathophysiological biochemical processes and consequently lead to neuronal death. Tizanidine (Sandoz compound DS 103–282, 5-chloro-4,2 (2-imidazolin-2-yl-amino)-2,1,3-benzothiazol hydrochloride) is a selective α 2 adrenoreceptor agonist which shows its effect by stimulating presynaptic α 2 adrenoreceptors in central ASPergic and GLUergic system by inhibiting aspartic acid and glutamic acid release. In this study, the effect of Tizanidine on reversible focal cerebral ischemia was evaluated. METHODS Cerebral blood flow to the left hemisphere of adult Sprague-Dawley rats ( n = 48) was temporarily interrupted by middle cerebral artery and bilateral common carotid artery occlusion for 3 hours in eight rats of each group. Tizanidine was given to each group of rats intraperitoneally before the ischemic insult, 2 hours after ischemia, right after the reperfusion, 2 h after reperfusion, and 4 hours after reperfusion; the animals survived for 24 hours after the reperfusion. After killing and triphenyltetrasoliumchloride staining of brain slices, infarction volumes and ratios of the brains were calculated and the results were compared with those of the control group. RESULTS Infarction volumes and infarction ratios of the Tizanidine group 1/2 hours before ischemia (143.7 ± 6.34 mm 3 and 10.1 ± 0.43%) and the Tizanidine group 2 hours after ischemia (145.6 ± 6.32 mm 3 and 10.3 ± 0.43%) were found to be significantly lower in favor of the Tizanidine groups when compared with those of the control group (173.9 ± 6.38 mm 3 and 12,4 ± 0.41%). Tizanidine is not effective if used just after reperfusion or later. CONCLUSION This study shows that Tizanidine pretreatment before the ischemic insult and the administration of the drug within the 2 hours after ischemia reduces ischemic damage significantly. Therefore, this drug can be used as a protective and therapeutic agent in ischemic diseases.

Glutamate, glutamine, and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in rats.

Even though the utilization of substrates alternative to glucose may play an important role in the survival of brain cells under ischemic conditions, evidence on changes in substrate selection by the adult brain in vivo during ischemic episodes remains very limited. This study investigates the utilization of glutamate, glutamine, and GABA as fuel by the neuronal and glial tricarboxylic acid cycles of both cerebral hemispheres after partially reversible focal cerebral ischemia (FCI). Right hemisphere infarct was induced in adult Long-Evans rats by permanent occlusion of the right middle cerebral artery and transitory occlusion of both common carotid arteries. (1,2-13C2) acetate was infused for 60 minutes in the right carotid artery immediately after carotid recirculation had been re-established (1-hour group) or 23 hours later (24-hour group). Extracts from both cerebral hemispheres were prepared and analyzed separately by 13C nuclear magnetic resonance and computer-assisted metabolic modeling. FCI decreased the oxidative metabolism of glucose in the brain in a time-dependent manner. Reduced glucose oxidation was compensated for by increased oxidations of (13C) glutamate and (13C) GABA in the astrocytes of the ipsilateral hemispheres of both groups. Increased oxidative metabolism of (13C) glutamine in the neurons was favored by increased activity of the neuronal pyruvate recycling system in the 24-hour group. Data were obtained consistent with time-dependent changes in the utilization of glutamate and GABA or glutamine as metabolic substrates for the glial or neuronal compartments of rat brain after FCI.