Neuroprotective effect of hexasulfobutylated C60 on rats subjected to focal cerebral ischemia

The effects of hexasulfobutylated C60 (FC4S), a free radical remover, on the total volume infarct size elicited by the damaging effects of focal cerebral ischemia were studied on Long-Evans rats in vivo. FC4S was administered intravenously either 15 min before middle cerebral artery (MCA) occlusion (pretreatment groups) or it was injected when the common carotid arteries clips were removed (treatment groups). FC4S did not alter the pH, blood gases, heart rate, or mean arterial blood pressure in either pretreatment or treatment groups of the rats. However, after administration of FC4S at dosages of 10 and 100 microg/kg, the total volume of infarction was significantly reduced in both pretreatment and treatment groups. In addition, after FC4S administration, the nitric oxide (NO) content in plasma was increased and the lactate dehydrogenase (LDH) levels was decreased. It is concluded that FC4S may be used as a neuroprotective agent on focal cerebral ischemia. The beneficial effects may be partly related to its antioxidant property and to the upregulation of NO production of the compound.     

The effect of desflurane on ameliorating cerebral infarction in rats subjected to focal cerebral ischemia-reperfusion injury

To obtain more information on the cerebral ischemia and reperfusion injury under desflurane anesthesia, we compared the infarct volume and lactate dehydrogenase (LDH) activity in rats subjected to focal cerebral ischemia during different concentration of desflurane anesthesia. Male Long-Evans rats weighing 270-350 g were anesthetized with desflurane in air at 1.0, 1.25 or 1.5 MAC whereas rats in the control group received intraperitoneal chloral hydrate (400 mg/kg) anesthesia. Cerebral infarction was induced by microsurgical procedures with ligation of the right middle cerebral artery (MCA) and clipping of the bilateral common carotid arteries (CCA) for 60 minutes. The rats were sacrificed 24 hours later, serial brain slices of 2mm thickness were taken and stained for the measurement of the infarct area. Cellular damage was evaluated by measuring the LDH level in the plasma. Desflurane (1.0, 1.25 or 1.5 MAC by inhalation) and chloral hydrate (400 mg/kg; ip.) did not produce any changes in pH, blood gases, heart rate or mean arterial blood pressure. In the rats subjected to focal cerebral ischemia, the volume of infarction was significantly less in the desflurane groups in all three different concentrations than in the chloral hydrate group. The changes of LDH activity in plasma also correlated with the result of the infarct volume. Our study suggests that desflurane may offer a neuroprotective effect such as decreased infarct volume after focal cerebral ischemia.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

Effect of combined therapy with hyperbaric oxygen and antioxidant on infarct volume after permanent focal cerebral ischemia

The aim of the present study was to evaluate the efficiency of combination of hyperbaric oxygen (HBO) and an antioxidant on permanent focal cerebral ischemia. Male Wistar rats underwent permanent middle cerebral artery occlusion (MCAO). Then, animals were randomly assigned to one of four groups: the control group (n=9) received no treatment, HBO group (n=9) was treated for 90 min at 2.5 absolute atmosphere for 3 days, the U-74389G group (n=8) received single U-74389G injection (3 mg/kg), the HBO + U-74389G group (n=8) received both HBO and U-74389G treatments. Treatments were initiated within the first 10 min after MCAO. After 3 days, the infarct volumes in rat brains were measured. The infarct ratios were 25.6+/-6.5 % for the control group, 21.9+/-6.4 % for the HBO group, 15.7+/-5.7 % for U-74389G group and 12.5+/-3.8 % for HBO + U74389G group. The infarct volumes were significantly reduced in rats treated with U-74389G (p<0.05) and combination therapy (p<0.05). HBO failed to reduce infarct volume significantly. We concluded that 1) U-74389G is more beneficial than HBO on permanent MCAO in rats, and 2) a combined therapy failed to significantly improve infarct volume more than either single treatment.     

Effects of 17beta-estradiol on blood-brain barrier disruption in focal ischemia during GABA(A) receptor inhibition.

We performed this study to determine whether gamma-aminobutyric acid (GABA(A)) receptor inhibition could reverse the effect of 17beta-estradiol on blood-brain barrier (BBB) disruption in focal cerebral ischemia. Young ovariectomized rats were implanted with a 500 microg 17beta-estradiol 21-day release pellet or with a vehicle pellet 21 days before the experiments. Forty-five minutes after middle cerebral artery (MCA) occlusion, half of each group was infused with bicuculline (a GABA(A) receptor antagonist) 1 mg/kg/min for 2 min followed by 0.1 mg/kg/min up to the end of experiments. The other half was infused with the same volume of normal saline. The transfer coefficient (Ki) of 14C-alpha-aminoisobutyric acid and the volume of 3H-dextran distribution (70,000 Daltons) were determined to measure the degree of BBB disruption one hour after MCA occlusion. In the control vehicle-treated animals, the Ki in the ischemic cortex (7.2 +/- 2.6 microl/g/min) was higher than in the contralateral cortex (2.5 +/- 1.4 microl/g/min). After bicuculline infusion, the Ki in the ischemic cortex increased (10.6 +/- 5.4 microl/g/min) although the increase was not statistically significant. In the 17beta-estradiol treated animals, the Ki in the ischemic cortex (3.8 +/- 1.6 microl/g/min) was lower than control vehicle-treated rats. With bicuculline infusion, the Ki in the ischemic cortex (14.5 +/- 6.8 microl/g/min) was markedly increased. In the non-ischemic cortex, there was no significant difference in Ki among the experimental groups. The volume of dextran distribution was not significantly different between the experimental groups in the ischemic or non-ischemic cortex. Our data suggests that part of the reason for the decreased BBB disruption in the focal ischemic area after 17beta-estradiol treatment could be due to the interaction between GABA(A) receptors and 17beta-estradiol.     

Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats

Free radicals have been implicated in neuronal injury during ischemia reperfusion in stroke. Trans resveratrol, a potent antioxidant, polyphenolic compound found in grapes and wines has recently been shown to have neuroprotective activity against oxidative stress in in vitro studies. In the present study the effect of chronic treatment of trans resveratrol was evaluated in focal ischemia induced by middle cerebral artery [MCA] occlusion in rats. Male Wistar rats were pretreated with trans resveratrol 20 mg/kg i.p. for 21 days and were subjected to focal ischemia by occlusion of MCA using intraluminal thread. After two hours of MCA occlusion reperfusion was allowed by retracting the thread. Animals were assessed for motor performance after 24 hours and subsequently rats were sacrificed for estimation of markers of oxidative stress [malondialdehyde [MDA] and reduced glutathione] and for evaluation of volume of infarction. Control group received vehicle and similar protocol was followed. Significant motor impairment, with elevated levels of MDA and reduced glutathione was observed in the vehicle treated MCA occluded rats. Treatment with trans resveratrol prevented motor impairment, rise in levels of MDA and reduced glutathione and also significantly decreased the volume of infarct as compared to control. The study provides first evidence of effectiveness of trans resveratrol in focal ischemia most probably by virtue of its antioxidant property.     

Early ischemic preconditioning against focal transient and permanent brain ischemia in rats: role of collateral circulation

We hypothesize that early ischemic preconditioning (IPC) can afford protection against focal brief and prolonged cerebral ischemia with subsequent reperfusion as well as permanent brain ischemia in rats by amelioration of regional cerebral blood flow. Adult male Wistar rats (n=97) were subjected to transient (30 and 60 minutes) and permanent middle cerebral artery (MCA) occlusion. IPC protocol consisted of two episodes of 5-min common carotid artery occlusion + 5-min reperfusion prior to test ischemia either followed by 48 hours of reperfusion or not. Triphenyltetrazolium chloride and Evans blue were used for delineation of infarct size and anatomical area at risk (comprises ischemic penumbra and ischemic core), respectively. Blood flow in the MCA vascular bed was measured with use of Doppler ultrasound. The IPC resulted in significant infarct size limitation in both transient and permanent MCA occlusion. Importantly, IPC caused significant reduction of area at risk after 30 min of focal ischemia as compared to controls [med(min-max) 11.4% (3.59-2 0.35%) vs. 2.47% (0.8-9.31%), p = 0.018] but it failed to influence area at risk after 5 min of ischemia [med(min-max) 7.61% (6.32-10.87%) vs. 8.2% (4.87-9.65%), p > 0.05]. No differences in blood flow were found between IPC and control groups using Doppler ultrasound. This is suggestive of the fact that IPC does not really influence blood flow in the large cerebral arteries such as MCA but it might have some effect on smaller arteries. It seems that, along with well established cytoprotective effects of IPC, IPC-mediated reduction of area at risk by means of improvement in local cerebral blood flow may contribute to infarct size limitation after focal transient and permanent brain ischemia in rats.     

Cinnamophilin reduces oxidative damage and protects against transient focal cerebral ischemia in mice

Acute neuroprotective effects of cinnamophilin (CINN; (8R, 8'S)-4, 4'-dihydroxy-3, 3'-dimethoxy-7-oxo-8, 8'-neolignan), a novel antioxidant and free radical scavenger, were studied in a mouse model of transient middle cerebral artery (MCA) occlusion. CINN was administered intraperitoneally either 15 min before (pretreatment) or 2 h after the onset of MCA occlusion (postischemic treatment). Relative to vehicle-treated controls, animals pretreated with CINN, at 20-80 mg/kg, had significant reductions in brain infarction by 33-46% and improvements in neurobehavioral outcome. Postischemic administration with CINN (80 mg/kg) also significantly reduced brain infarction by 43% and ameliorated neurobehavioral deficits. Additionally, CINN administration significantly attenuated in situ accumulation of superoxide anions (O2-) in the boundary zones of infarct at 4 h after reperfusion. Consequently, CINN-treated animals exhibited significantly decreased levels of oxidative damage, as assessed by immunopositive reactions for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxynonenal (4-HNE), and the resultant inflammatory reactions at 24 h post-insult. It is concluded that CINN effectively reduced brain infarction and improved neurobehavioral outcome following a short-term recovery period after severe transient focal cerebral ischemia in mice. The finding of a decreased extent of reactive oxygen species and oxidative damage observed with CINN treatment highlights that its antioxidant and radical scavenging ability is contributory.     

Effects of chronic L-NAME treatment on rat focal cerebral ischemia and cerebral vasoreactivity.

Nitric oxide has been shown to be involved in the regulation of cerebral blood flow and the consequences of cerebral ischemia. Short-term inhibition of its synthesis induces hypertension and increases the cortical infarct volume in focal ischemia. Our purpose was to investigate the influence of the long-term inhibition of nitric oxide synthase on infarct volume due to middle cerebral artery (MCA) occlusion and on the reactivity of cerebral arteries. Sprague Dawley rats were given N(omega)-nitro-L-arginine methyl ester (L-NAME) for 2 or 6 weeks and compared to untreated normotensive rats and untreated spontaneously hypertensive rats (SHRs). Brain nitric oxide synthase activity was measured by the 14C-L-arginine assay. Arterial blood pressure was measured in each group. Independently, the reactivity of MCA trees was studied in vitro by a perfusion technique. Cortical infarct volume was not significantly modified by either 2-week or 6-week L-NAME treatment, despite induced hypertension, whereas it was significantly higher in SHRs than in normotensive rats. The reactivity of the MCA tree was significantly affected by the treatment with a clearcut time-dependency. Compared to normotensive controls, contractility to noradrenaline and serotonin was reduced, more severely at 6 weeks, and while dilatation to acetylcholine and nitroprusside was moderately reduced at 6 weeks, dilatation to papaverine was then increased. A major difference of treated animals compared to SHRs was the decreased response to 5-hydroxytryptamine. We conclude that infarct expansion may be limited in treated animals by a progressive reduction in cerebral artery response to vasoconstrictory neurotransmitters, concomitant with augmented non-guanylate cyclase dilator responses (cf. papaverine) and some recovery of dilatation to acetylcholine.     

Nimodipine inhibits calcium-independent nitric oxide synthase activity in transient focal cerebral ischemia rats and cultured mouse astroglial cells

The effect of nimodipine on nitric oxide synthase (NOS) activities in brains in transient focal cerebral ischemia rats, in cultured mouse neurons and astroglial cells and bovine brain capillary endothelial cells (BCECs) was investigated. The administration of nimodipine (3 mg.kg(-1), p.o., twice a day, for 3 days) before middle cerebral artery (MCA) occlusion significantly reduced infarct size, decreased nitrite/nitrate (NOx) content and inhibited Ca2+-independent NOS activity in the infarct area. Nimodipine inhibited the Ca2+-independent NOS activity induced by lipopolysaccharide (LPS) + tumor necrosis factor alpha (TNF alpha) in mouse astroglial cells with an IC50 value of 0.036+/-0.003 mM and Ca2+-dependent NOS activity in mouse neurons with an IC50 value of 0.047+/-0.003 mM, but did not affect Ca2+-dependent NOS activity in BCECs. The inhibition of Ca2+-independent NOS activity by nimodipine in astroglial cells was competitive with respect to L-arginine. Nimodipine also inhibited the induction of Ca2+-independent NOS activity in vitro. These results suggest that nimodipine in addition to its cerebral vasodilating effect may protect brain from ischemic neuronal damage through modifying NOS activity.     

Neuroprotective effects of hydroxyethylpuerarin against focal cerebral ischemia-reperfusion in rats

Our present study was performed to investigate whether hydroxyethylpuerarin (HEP) has a neuroprotective effect on brain injury after focal cerebral ischemia/reperfusion by middle cerebral artery occlusion (MCAO) in adult male Wistar rats. Animals were subjected to one hour of middle cerebral artery occlusion and 48 hours of reperfusion with the pretreatment of drugs (HEP 15, 30, 60 mg/ kg or nimodipine 0.4 mg/kg i.v.) or vehicle. The behavioral tests were used to evaluate the damage to central nervous system. The percentage of brain infarct area was assessed in the brain slices stained with 2% solution of 2, 3, 5-triphenyl tetrazolium chloride (TTC). The pathologic histological changes were observed by H&E staining and the occurrence of apoptosis was determined by flow cytometry. The results showed that pretreatment with HEP at doses of 15, 30, 60 mg/kg exhibited significant neuroprotective effects on rats against focal cerebral ischemia-reperfusion injury by markedly decreasing neurological deficit scores and the percentage of infarct area, reducing necrosis and apoptosis of neurons. All these findings suggest that HEP might provide neuroprotection against focal cerebral ischemia/reperfusion injury probably through its antioxidant and anti-inflammatory property.     

TLR2 has a detrimental role in mouse transient focal cerebral ischemia

A significant up-regulation of Toll-like-receptor (TLR) mRNAs between 3 and 48 h reperfusion time after induction of transient focal cerebral ischemia for 1h was revealed by applying global gene expression profiling in postischemic mouse brains. Compared to TLR4 and TLR9, TLR2 proved to be the most significantly up-regulated TLR in the ipsilateral brain hemisphere. TLR2-protein was found to be expressed mainly in microglia in the postischemic brain tissue, but also in selected endothelial cells, neurons, and astrocytes. Additionally, TLR2-related genes with pro-inflammatory and pro-apoptotic capabilities were induced. Therefore we hypothesized that TLR2-signaling could exacerbate the primary brain damage after ischemia. Two days after induction of transient focal cerebral ischemia (1h), we found a significant decrease of the infarct volume in TLR2 deficient mice compared to wild type mice (75+/-5 vs. 42+/-7 mm(3)). We conclude that TLR2 up-regulation and TLR2-signaling are important events in focal cerebral ischemia and contribute to the deterioration of ischemic damage.     

Reversibility of Nimodipine Binding to Brain in Transient Cerebral Ischemia

Using autoradiography, we have measured the in vivo binding of [3H]nimodipine to brain in a rat model of reversible cerebral ischemia. Ischemia was induced by simultaneous occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery by microaneurysm clips. Rats were studied after 15 min of ischemia (ischemic group) or after 45 min of reperfusion following 15 min of ischemia (reperfused group). Regional cerebral blood flow (CBF) was determined autoradiographically using [14C]iodoantipyrine in both ischemic (n = 6) and reperfused (n = 6) groups. During ischemia blood flow in the territory of the MCA was depressed and recovered to normal only in the distal territory of the MCA following reperfusion. [3H]Nimodipine binding in the ischemic group (n = 12) was elevated in ischemic brain regions and declined significantly (p < 0.01) in these regions in the reperfused group (n = 11). The ratio of the volume of cortex showing increased binding to the total volume of the forebrain was 0.113 +/- 0.025 (mean +/- SD) in the ischemic group and declined to 0.080 +/- 0.027 following reperfusion (p < 0.005). In general, infarct was only observed in regions showing persistent elevation of nimodipine binding following reperfusion as determined by histology performed in a separate group of rats (n = 8) after 24 h of reperfusion. We conclude that increased nimodipine binding to ischemic tissue is initially reversible with prompt reestablishment of CBF and is a sensitive indicator of early and reversible ischemia-induced cerebral dysfunction.     

Characterization of a new double-filament model of focal cerebral ischemia in heme oxygenase-2-deficient mice

Variations in vascular anatomy in knockout mouse strains can influence infarct volume after middle cerebral artery (MCA) occlusion (MCAO). In wild-type (WT) and heme oxygenase-2 gene-deleted (HO2-/-) mice, infarcts were not reproducibly achieved with the standard intraluminal filament technique. The present study characterizes a double-filament model of MCAO, which was developed to produce consistent infarcts in both WT and HO2-/- mice. Diameters of most cerebral arteries were similar in WT and HO2-/- mice, although the posterior communicating artery size was variable. In halothane-anesthetized mice, two 6-0 monofilaments with blunted tips were inserted into the left internal carotid artery 6.0 and 4.5 mm past the pterygopalatine artery junction to reside distal and proximal to the origin of the MCA. The tissue "volume at risk" determined by brief dye perfusion in WT (59 +/- 2% of hemisphere; +/-SE) was similar to HO2-/- (62 +/- 4%). The volume of tissue with cerebral blood flow <50 ml.min(-1).100 g(-1) was similar in WT (35 +/- 9%) and HO2-/- (36 +/- 11%) during MCAO and at 3 h of reperfusion (<2%). After 1 h MCAO, infarct volume was greater in HO2-/- (44 +/- 6%) than WT (25 +/- 3%). After increasing MCAO duration to 2 h, the difference between HO2-/- (47 +/- 4%) and WT (36 +/- 3%) diminished, but infarct volume remained substantially less than the volume at risk. Infusion of tin protoporphyrin IX, an HO inhibitor, during reperfusion after 1 h MCAO increased infarct volume in WT but not significantly in HO2-/- mice, although infarct volume remained less than the volume at risk. Thus greater infarct volume in HO2-/- mice is not attributable to a greater volume at risk, lower intraischemic blood flow, or poor reflow, but rather to a neuroprotective effect of HO2 activity. The double-filament model may be of use as an alternative in other murine knockout strains in which the standard filament model does not yield consistent infarcts.     

SM-31900, a novel NMDA receptor glycine-binding site antagonist,reduces infarct volume induced by permanent middle cerebral artery occlusion in spontaneously hypertensive rats

The purpose of this study was to investigate the effect of (3S)-7-chloro-3-[2-((1R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900), an antagonist with high selectivity and affinity for the NMDA receptor glycine-binding site, on the cerebral infarct volume in a permanent middle cerebral artery occlusion (MCAo) model, which was constructed by electrocoagulation of a unilateral middle cerebral artery distal to the olfactory tract using spontaneously hypertensive rats (SHRs). To investigate the dose-response characteristics and the therapeutic time window of SM-31900 in this MCAo model, we conducted three experiments, in which the administration of SM-31900 was started 5min (experiment I), 30min (experiment II), or 60min (experiment III) after MCAo, respectively. In all the studies, SM-31900 was administered by intravenous bolus injection followed by continuous intravenous infusion to obtain a steady-state level of this compound in blood immediately after its administration. The treatment with SM-31900 was continued until 24h after MCAo, at which time the cerebral infarct volume was measured. In experiment I, SM-31900 significantly reduced the infarct volume by 37% at a dosage of 0.38mg/kg bolus followed by 1.5mg/kg/h continuous infusion (0.38mg/kg+1.5mg/kg/h). In experiment II, the neuroprotective effect of SM-31900 was also significant, with a 25% reduction in infarct volume at a dosage of 0.38mg/kg+1.5mg/kg/h, and a 40% reduction at 1.5mg/kg+6.0mg/kg/h. Furthermore, even in experiment III, SM-31900 exerted a significant neuroprotective effect, with a 20% reduction at 1.5mg/kg+6.0mg/kg/h. These studies revealed that SM-31900 can exert a neuroprotective effect when it is administered up to at least 60min after the onset of ischemia in the MCAo model, an animal model of stroke, indicating that SM-31900 is a good candidate for treating acute brain ischemia.     

Effect of endothelin antagonist (TAK-044) on cerebral ischemic volume, oxidative stress markers and neurobehavioral parameters in the middle cerebral artery occlusion model of stroke in rats

Stroke causes brain injury in millions of people world wide each year. Despite the enormity of problem, currently there is no established therapy, which can restore the blood flow at infracted area and also improve the neurological deficit. The present study was carried out to investigate the effect of an endothelin antagonist (TAK-044) in middle cerebral artery (MCA) occlusion model of acute ischemic stroke in rats. Male Wistar rats were pretreated with TAK-044 (5 mg/kg, i.p.) for 7 days and thereafter subjected to focal ischemia by occlusion of MCA using intraluminal thread for two hours. 30 min after reperfusion the animals were subjected to diffusion-weighted imaging (DWI) for assessment of protective effect. Twenty-four hours later the motor performance was tested and subsequently the animals were sacrificed for estimation of markers of oxidative stress; malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD). Control group received vehicle (saline) and similar experimental protocol was followed. In the TAK-044 pretreated group, percent hemispheric lesion area (% HLA) in DWI was significantly attenuated 17.5 +/- 0.5% as compared to control group 61.2 +/- 5.9%. Significant motor impairment, with significant elevated levels of MDA, decrease in GSH and SOD were observed in the vehicle treated MCA occluded rats. Pretreatment with TAK-044 prevented the motor impairment and significantly reversed the changes in markers of oxidative stress (MDA, GSH and SOD). In addition to well-known vasodilatory effect, TAK-044 has recently been documented to have antioxidant and anti-inflammatory properties. These effects can contribute to the protection afforded by TAK-044 in the present study.     

Transient middle cerebral artery occlusion causes different structural, mechanical, and myogenic alterations in normotensive and hypertensive rats

Transient focal cerebral ischemia in the rat alters vessel properties, and spontaneously hypertensive rats (SHR) show a poorer outcome after ischemia. In the present study we examined the role of hypertension on vessel properties after ischemia-reperfusion. The right middle cerebral artery (MCA) was occluded (90 min) and reperfused (24 h) in SHR (n = 12) and Wistar-Kyoto rats (WKY; n = 11). Sham-operated rats (SHR, n = 10; WKY, n = 10) were used as controls. The structural, mechanical, and myogenic properties of the MCA were assessed by pressure myography. Nuclei distribution and elastin content and organization were analyzed by confocal microscopy. Infarct volume was larger in SHR than in WKY rats. Ischemia-reperfusion induced adventitial hypertrophy associated with an increase in the total number of adventitial cells. In addition, fenestrae area and arterial distensibility increased and myogenic tone decreased in the MCA of WKY rats after ischemia-reperfusion. Hypertension per se induced hypertrophic inward remodeling. Ischemia-reperfusion decreased the cross-sectional area of the MCA in SHR, without significant changes in distensibility, despite an increase in fenestrae area. In addition, MCA myogenic properties were not altered after ischemia-reperfusion in SHR. Our results indicate that in normotensive rats, MCA develops a compensatory mechanism (i.e., enhanced distensibility and decreased myogenic tone) that counteracts the effect of ischemia-reperfusion and ensures correct cerebral irrigation. These compensatory mechanisms are lost in hypertension, thereby explaining, at least in part, the greater infarct volume observed in SHR.     

Acute but not chronic tempol treatment increases ischemic and reperfusion ventricular arrhythmias in open-chest rats

The effect of the chronic and acute antioxidant tempol (superoxide dismutase mimetic) treatment on cardiac ischemic tolerance was investigated in adult male Wistar rats. The first experimental group was given tempol (1 mM) in drinking water for three weeks, the second group received tempol (100 mg/kg, i.v.) 10 min before test ischemia, and control rats received the same volume of solvent. Anesthetized open-chest animals (pentobarbitone 60 mg/kg, i.p.) were subjected to 20-min coronary artery occlusion and 3-h reperfusion for infarct size determination. Ventricular arrhythmias were monitored during ischemia and at the beginning (5 min) of reperfusion. Acute tempol administration shifted the time profile of ischemic arrhythmias to the later phase and significantly increased the number of ischemic and reperfusion premature ventricular complexes, respectively (504+/-127 and 84+/-21) as compared with the chronically treated group (218+/-36 and 47+/-7) or controls (197+/-26 and 31+/-7). Acute tempol-treated rats exhibited a tendency to decrease infarct size (P = 0.087). The mechanism of proarrhythmic tempol action during ischemia and reperfusion remains to be elucidated.     

Bradykinin elicits "second window" myocardial protection in rat heart through an NO-dependent mechanism

Bradykinin is an important endogenous mediator exerting acute protective effects in the ischemic myocardium. The aims of this study were to investigate whether exogenously administered bradykinin could evoke delayed myocardial protection and to determine whether any protection observed might be dependent on nitric oxide (NO) generation. Conscious rats received bradykinin (40 microg/kg iv) or saline, preceded 15-20 min earlier by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg ip) or saline. Twenty-four hours later, hearts were Langendorff perfused and subjected to 35 min of regional ischemia and 120 min of reperfusion. Infarct size was assessed using tetrazolium staining and expressed as a percentage of the risk zone. Bradykinin pretreatment reduced the infarct-to-risk ratio from 53.5 +/- 3.2% to 29.1 +/- 4.7% (P < 0.01). The administration of L-NAME before bradykinin abrogated the delayed protection (infarct size 52.3 +/- 5.0%) but alone did not influence infarct size (53.5 +/- 4.8%). These results are the first to demonstrate that bradykinin can evoke a delayed ("second window") enhancement of myocardial tolerance to ischemia, an action that is dependent on the early generation of NO.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.