doi: 10.1161/hs0901.095640
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Stroke. 2001;32:2157.)
© 2001 American Heart Association, Inc.
Original Contributions |
Combination of a Free Radical Scavenger and Heparin Reduces Cerebral Hemorrhage After Heparin Treatment in a Rabbit Middle Cerebral Artery Occlusion Model
From the Department of Pharmacology, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan.
Correspondence to Dr Kazuo Umemura, Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192, Japan. E-mail umemura{at}hama-med.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Background and Purpose—— We sought to investigate the effects of EPC-K1, a free radical scavenger, on reducing heparin-produced cerebral hemorrhage in a rabbit model of middle cerebral artery (MCA) photothrombosis and to investigate whether the combination of EPC-K1 and heparin enhances neuroprotection from cerebral ischemic damage.
Methods—— In the heparin-alone group (n=8), heparin was administered intravenously for 24 hours, starting from 3 hours after MCA occlusion. In the EPC-K1-alone group (n=8), EPC-K1 was administered as a bolus injection (10 mg/kg) twice at 3 and 6 hours after MCA occlusion. In the combination group (n=8), EPC-K1 and heparin both were administered as in the single-drug procedures. In the vehicle group (n=10), saline were infused for 24 hours.
Results—— Heparin prolonged activated partial thromboplastin time by 
3 times that of control animals. In the heparin-treated animals, the hemorrhage size was significantly increased (P<0.0001) and neurological symptoms were significantly worse (P<0.01) than in control animals at 48 hours. The combination of EPC-K1 and heparin dramatically reduced heparin-produced cerebral hemorrhage (P<0.0001), with a significant reduction in infarct volume (reduction by 63.2% and 57.2% of heparin-alone and control animals, respectively, P<0.0001) and a significant improvement in neurological symptoms (P<0.01 versus heparin-alone and control animals, respectively).
Conclusions—— These data indicate that free radical formation may play a key role in intracerebral hemorrhage exacerbated by heparin treatment and that the combination of a free radical scavenger and heparin augmented neuroprotection from acute brain ischemia. The results of the present study may suggest a potential clinical approach for the treatment of acute stroke.
Key Words: cerebral ischemia • free radicals • heparin • intracerebral hemorrhage • middle cerebral artery occlusion • rabbits
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Intracerebral hemorrhagic transformations are feared events that may follow therapy with antithrombotic and thrombolytic agents in acute stroke, so the high-dose use of these agents has been limited.1,2 The merit of antithrombotic agents in clinical use depends on a balance between the benefits of a reduction in infarct volume and the risks of cerebral hemorrhage.
Published evidence indicates that matrix metalloproteinases (MMPs) increase in primates with hemorrhagic transformation after cerebral ischemia-reperfusion injury,3 and the activated forms of MMPs appeared after the blood-brain barrier opening4 and intracerebral hemorrhage.5 On the other hand, overexpression of superoxide dismutase in transgenic mice has been observed to reduce MMP expression after brain trauma.6 Recently, 2 spin-trap agents and metalloproteinase inhibition have been found to reduce thrombolysis-induced hemorrhage after thromboembolic stroke.7,8 These results together suggest a possible association of free radicals with intracerebral hemorrhage after heparin treatment.
Based on photothrombotic technique, we developed a new model of intracerebral hemorrhage induced by antithrombotic agents after middle cerebral artery (MCA) thrombotic occlusion in rabbits.9,10 In this model, spontaneous reperfusion of the MCA after the thrombotic occlusion following cyclic flow reductions was important.10,11 The rabbit was chosen because it is often used for various models of intracranial hemorrhage.12
In the present study, therefore, we wanted to use the cerebral hemorrhage model produced with heparin to investigate whether the addition of EPC-K1, a free radical scavenger, reduces the hemorrhage by heparin and, if so, whether such combination treatment enhances the neuroprotective efficacy regarding cerebral ischemic damage. EPC-K1 is a hydroxyl radical scavenger, and its chemical structure is combined with vitamins C and E by phosphate.13
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The experimental protocol was approved by the Hamamatsu University School of Medicine Committee on Ethics of Animal Experimentation, and extra care was taken to avoid causing animal suffering.
Animal Preparation
Male Japanese White rabbits weighting 2.0 to 3.0 kg were anesthetized with 1% to 2% isoflurane (Dainihon Pharmaceutical Co) in a mixture of 30% O2 and 70% N2O with the use of a face mask. The animals were restrained in the right lateral position with spontaneous respiration, and body temperatures were maintained at 38°C with a heating pad (K-module model K-20; American Pharmaseal) during the operation. A plastic catheter was placed in the right femoral vein for the infusion of agents or saline. Another catheter filled with a minimal dose of heparinized saline was cannulated in the ipsilateral femoral artery for blood sampling and connected to a pressure transducer (Nihon Konden); arterial blood pressure and heart rate were recorded continuously throughout the surgical procedure. Arterial blood gases were monitored using a gas analyzer (model 860; Ciba-Corning) just before and 1 hour after the start of photoirradiation.
MCA Thrombotic Occlusion
The photothrombotic occlusion of the MCA was performed in a similar manner, as described previously by Umemura and colleagues.9,14 In brief, after removal of the eyeball and the temporalis muscle with a bipolar electric coagulator (model 80-1160; Valley Forge Scientific Corp) and a thermoknife, an 8-mm-diameter oval window was opened with a dental drill (model PAL-7; Morita). Under the operating microscopy, the main trunk of the MCA was observed without cutting the dura mater. Photoirradiation with green light (wavelength 540 nm) was achieved by using a xenon lamp (model L4887; Hamamatsu Photonics) with a heat-absorbing filter and a green filter, whereas Watson and colleagues used laser irradiation with either a 514.5-nm argon beam15 or a 562-nm argon laser-activated dye laser beam.16 The machine of Watson and colleagues is more efficient in causing a photochemical reaction between rose bengal and light irradiation than is our machine. To occlude a longer length of the rabbit MCA, a 3-mm-diameter optic fiber mounted on a micromanipulator was used. The surrounding tissue was shielded with shims to irradiate only the MCA. The head of the optic fiber was placed on the origin of the MCA passing over the olfactory tract, providing an irradiation dose of 0.170 W/cm2. The probe of a pulsed Doppler flowmeter (PVD-20; Crystal Biotech) was positioned on the MCA segment distal to the irradiated area to measure the MCA blood flow. When steady cerebral blood flow was obtained, rose bengal (10 mg/kg body wt for 3 minutes) was injected through a peripheral ear vein. At the same time, photoirradiation was started for 60 minutes. The local blood flow in MCA was continuously monitored for 1 hour after rose bengal injection. After the surgical wounds were closed, the animals were allowed to recover from anesthesia.
The following parameters were measured for the evaluation of MCA blood flow: (1) occlusion time, defined as the time from the start of the rose bengal injection to the cessation of blood (as indicated by the flow monitor) in the MCA, (2) total reperfusion time, represented as the amount of time when the vessel was open from the start of the rose bengal injection until 1 hour after the start of photoirradiation, and (3) first reperfusion time, defined as the time when the vessel was first reopened after the thrombotic occlusion.
In a separate experiment with 2 rabbits (occlusion of the MCA was performed as described), plasma rose bengal concentrations were determined spectrophotometrically (Ultrospec-2001; Hitachi Co) at a 550-nm wavelength after appropriate dilution with water. The rose bengal blood concentrations in the 2 rabbits were 193.1 and 208.8 µmol/L at 3 minutes, 90.9 and 75.1 µmol/L at 10 minutes, 49.9 and 40.0 µmol/L at 30 minutes, and 37.8 and 33.4 µmol/L at 60 minutes after the rose bengal injection (10 mg/kg).
Measurement of Temperature at the Site of the Irradiated Tissue Near the MCA
To verify that irradiation or the photochemical reaction did not heat the irradiated tissue, in 3 animals the temperature at the site of the irradiated tissue was measured continuously with an implanted digital thermometer probe (0.5 mm diameter; Inter Medical). The digital thermometer probe was touched directly to the MCA and was at the irradiation center (it is impossible to measure the temperature inside the rabbit’s MCA).
Administration of EPC-K1 and Heparin
EPC-K1 and heparin were diluted with saline. All bolus injections were made at a volume of 1 mL/kg, and heparin infusion was made at a delivery rate of 0.5 mL · kg-1 · h-1. In the heparin-alone group, heparin was administered as an intravenous bolus injection of 100 IU/kg followed by a 75 IU · kg-1 · h-1 continuous infusion for 24 hours, beginning 3 hours after the start of photoirradiation in 8 animals. In EPC-K1-alone group (n=8), EPC-K1 was administered intravenously as a bolus injection of 10 mg/kg twice at 3 and 6 hours after the start of photoirradiation. In the combination of EPC-K1 and heparin group (n=8), EPC-K1 and heparin both were administered as in the single-drug procedures. In the vehicle-treated group (control), 10 animals were infused continuously with saline beginning 3 hours for 24 hours.
Drugs
Heparin (1000 IU/mL) was purchased from Hoechst Pharmaceutical Co. Rose bengal was purchased from Wako Chemical Co. EPC-K1 (potassium ({(5R)-5-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-2-oxo-2,5-dihydro-3-furanyl}oxy)(oxo){[2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydro-2H-chromen-6-yl]oxy}phosphoranolate) was obtained from Toyama Chemical Co.
Analysis of Activated Partial Thromboplastin Time
Analysis of activated partial thromboplastin time (aPTT) was performed using an automatic Coagulometer KC4A (Heinrich Amelung) as previously described.9 In all groups, aPTT was measured before photoirradiation and 24 hours after drug administration.
Examination of Neurological Symptoms
Neurological symptoms of each animal were examined blindly 48 hours after photothrombosis. In the wry-neck test, the animal was placed in a cage and the torsion of the neck was observed. Behavior was scored, with 0 indicating normal and 1 indicating twisting of the neck. In the righting reflex test, the animal was placed on its back, and scored as follows: 0, righted within 1 second; 1, righted within 5 seconds; or 2, did not right within 5 seconds. Regarding paw dysfunction, the forepaw or hindpaw was pulled toward the body. The time to reextend the paw was scored as follows: 0, achieved within 1 second; 1, achieved within 5 seconds; or 2, not achieved within 5 seconds. In the postural reflex test, the animal was pushed in the contralateral direction and scored as follows: 0, normal; 1, reduced resistance to lateral push; or 2, fell down on the contralateral side. In the circling test, the animal was placed on the ground; 1 point was given when the animal circled with touching the body, and 2 points were given when the animal circled without touching the body. Each score was summed and represented the neurological score (0 to 11). In the wry-neck test, shoulder adduction was observed. In paw dysfunction, mild wrist flexion or full flexion of the wrist and elbow was observed. In the righting reflex test, the postural reflex test, and the circling test, hemiparesis and obtundation of animals were observed.
Determination of Cerebral Hemorrhage Volume
After measurement of the other variables at 48 hours after photothrombosis, animals were intravenously injected with pentobarbital sodium and then immediately perfused transcardially with normal saline. Their cerebrums were rapidly isolated. Twelve consecutive coronal sections that were 1 mm thick were cut from each of cerebrums using a slicing apparatus (RBM-7000C; Activatinal Systems Inc). All coronal sections were immediately photographed. The red and brown areas in the aspects of the sections were defined as hemorrhage. The size of cerebral hemorrhage was measured using a computerized image analysis system (NIH Image 1.62 Program; Internet).
Determination of Cerebral Infarct Volume
The brain slices were immersed for 30 minutes in a 1% solution of triphenyltetrazolium chloride at 37°C and then fixed with buffered formaldehyde (pH 7.2) for 24 hours. Photographs of each section were taken again after staining with triphenyltetrazolium chloride, and the size of cerebral infarction was measured with the computerized image analysis system NIH Image. The unstained and white softening areas on each section were considered to be the areas of cerebral ischemic damage.
Statistical Analysis
Data are represented as mean±SD. Physiological variables, parameters of cerebral blood flow, aPTT, size of cerebral hemorrhage, and area of cerebral damage were analyzed statistically by 1-way ANOVA and Fisher’s PLSD test. Scores in neurological deficits were analyzed by Kruskal-Wallis test followed by the Mann-Whitney U test. P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Temperature at the Site of the Irradiated Tissue Near the MCA
During the low-intensity irradiation period, the temperature at the site of the irradiated tissue near the MCA was not increased (Figure 1).
|
Physiological Parameters
Table 1 summarizes the values of physiological variables obtained at pretreatment and 1 hour after the start of photoirradiation in each group. No significant differences were noted among the 4 groups in these parameters after the surgical procedure and the photothrombotic MCA occlusion.
|
Blood Flow in the MCA
The blood flow in the MCA was occluded completely by the formation of platelet-rich thrombus at 10.8±2.2 minutes after the rose bengal injection. There was no significant difference in the time to occlusion after the start of photoirradiation among the 4 groups, and this revealed that the degree of damage to the MCA was the same (Table 2). Figure 2 shows a typical recording of cyclic flow reductions in the MCA. Spontaneous reperfusion of the occluded MCA after cyclic flow reductions was observed within 1 hour in 85.3% of animals (29 of 34). Reperfusion and reocclusion in the MCA, the first reperfusion time, and the total vascular patency time during the 1-hour observation period did not show significant differences among the 4 groups.
|
|
aPTT Values
The baseline aPTT values were similar among the 4 groups (Table 2). The administration of saline or EPC-K1 did not affect aPTT values. In the heparin-alone group or the combination of EPC-K1 and heparin group, aPTT was markedly prolonged at 24 hours after the infusion of heparin by 3 times compared with the control group. EPC-K1 did not affect aPTT prolonged with heparin.
Neurological Symptoms
The median neurological score at 48 hours was 4.5 in the vehicle group (Figure 3). The administration of heparin, beginning 3 hours after photothrombosis, significantly worsened neurological symptoms (P<0.01) compared with the control group. On the contrary, the combination treatment of EPC-K1 and heparin significantly improved the neurological score to 0.5 (P<0.01 versus heparin alone and control, respectively). There was no significant difference in neurological scores between the EPC-K1-alone animals and the vehicle-treated animals.
|
Cerebral Hemorrhage Size and Cerebral Infarct Volume
Figure 4 shows typical photographs of intracerebral hemorrhage and cerebral infarction in each group. In most of the animals, hemorrhage was mainly observed in the basal ganglia. The administration of heparin significantly increased the size of cerebral hemorrhage (79.4±43.2 versus 23.4±17.3 mm3 in the control group, P<0.0001) (Figures 4 and 5). The total infarct volume in the heparin-alone animals also tended to be larger than that of control group (304.5±64.9 versus 261.8±40.1 mm3 in the control group, P=0.06) (Figure 6). EPC-K1 alone did not affect the hemorrhagic size (P=0.39 versus control) and significantly decreased the infarct volumes of total and basal ganglia by 29.1% (P<0.01 versus control) and 31.3% (P<0.01 versus control), respectively (Figure 6). The combination treatment with EPC-K1 and heparin dramatically reduced heparin-produced hemorrhage (P<0.0001 versus heparin alone) (Figures 4 and 5). Consequently, the infarct volumes of the cortex (by 66.5% of heparin alone, P<0.01; by 60.0% of control, P<0.01) and of the basal ganglia (by 62.2% of heparin alone, P<0.0001; by 56.3% of control, P<0.0001) and the total volume (by 63.2% of heparin alone, P<0.0001; by 57.2% of control, P<0.0001) were significantly reduced (Figure 6). Infarct volume in the combination of EPC-K1 and heparin group was significantly decreased by 39.6% (P<0.01) (Figure 6) compared with the EPC-K1-alone group.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, the formation of a platelet- and fibrin-rich thrombus in the MCA was induced photochemically with rose bengal injection and green light from a xenon lamp irradiation system.17 The thrombotic occlusion of MCA caused by endothelial injury through singlet oxygen resulted in ischemic lesions in the brain tissues.17,18 The composition of the thrombus is different than the pure platelet thrombus induced by the laser irradiation.15,16 With use of the photothrombotic model in the rabbit MCA, the present study demonstrated that the administration of heparin initiated 3 hours after thrombotic occlusion of the MCA increased the size of intracerebral hemorrhage and resulted in worse neurological symptoms. EPC-K1, a hydroxyl radical scavenger, reduced the heparin-produced hemorrhage and thus decreased infarct volume and improved neurological symptoms. The results suggest that free radicals may play a key role in the development of intracerebral hemorrhage via heparin treatment in ischemic brain damage.
We recently developed and reported a model of cyclic flow reductions in the guinea pig11 and in the rabbit9,10 in which a 3-mm-diameter optic fiber was used. Up to now, we have used only a 3-mm-diameter optic fiber, and therefore, it is presently unclear to us whether the occlusion of a 3-mm segment of the MCA is the most appropriate length for the observation of cyclic flow reductions. We used 10 minutes of irradiation before the injection of rose bengal in our earlier study.19 In our recent reports, rose bengal injection and photoirradiation were started simultaneously.9–11 We have seen no difference in the formation of thrombus in the MCA and the cerebral infarct volume between the 2 approaches.
Sixty percent of patients recover MCA blood flow in the early phase of ischemic stroke.20 In our recent reports, animals that had more cyclic flow reductions had a significantly higher cerebral hemorrhage volume.10 Therefore, we sought to establish an MCA thrombotic occlusion model in which continued cyclic flow reductions are observed after reperfusion of the occluded MCA. This model is thought to readily induce cerebral hemorrhage by antithrombotic agents. In the preliminary studies, we examined various experimental conditions, including various doses of rose bengal and intensities of irradiation, and we measured the corresponding infarct volumes together with neurological deficits. For example, for rose bengal injection at 10 mg/kg with an irradiation intensity of 0.170 W/cm2 for 30 minutes, although the blood flow to the MCA was occluded at 10 minutes after rose bengal injection, the occluded MCA reperfused immediately after irradiation was stopped, which discontinued the cyclic flow reductions, provided small infarct volume, and did not allow assessment of drug efficacy. To maintain the cyclic flow reductions, irradiation was also continued after reperfusion of the occluded MCA. The low-intensity irradiation that we used did not increase the temperature at the site of the irradiated tissue near the MCA (Figure 1). Our data and that from Klaassen21 demonstrated that rose bengal also exists in the blood of the rabbit at 60 minutes after injection. On this basis, we used a low dose of rose bengal and longer but low-intensity irradiation time to produce a gradual vascular injury; under these conditions, cyclic flow reductions continued. As expected, heparin aggravated hemorrhage by 3.4-fold compared with the vehicle group in this model.
Clinical trials in patients with acute stroke showed that higher doses and delayed treatment of heparin were associated with increased intracerebral hemorrhage rates,1,2 leading to masking of its benefit. The dose limitations of heparin may also reduce its efficacy. The mechanisms of hemorrhage by heparin are still unclear. In the present study, heparin significantly increased intracerebral hemorrhage when administrated beginning 3 hours after MCA occlusion. Further, heparin induced hemorrhagic infarction, resulting in slightly, but not significantly, increased infarct volume. In the present study, heparin prolonged aPTT by 3 times compared with the vehicle group, which is close to the dose used clinically.22 However, heparin at the same dose, which was administered beginning 30 minutes after MCA occlusion, could reduce infarct volume without enhancement of cerebral hemorrhage.9 This suggests that the risk of hemorrhage with heparin is associated with the time from the onset of ischemia to the initiation of treatment, and cerebral hemorrhage due to heparin limits its neuroprotective effects regarding cerebral ischemic damage in this model. Previous reports showed that reperfusion injury may be associated with hemorrhage after brain ischemia.23 Early reperfusion was found in most of animals in this study, but the time of the first reperfusion and the total patency time of the MCA did not differ between heparin-treated animals and control animals during the 1-hour observation period. These results suggest that heparin-produced hemorrhage in this model may not be caused by early recanalization of the occluded MCA. The possible explanation for heparin-produced hemorrhage is that heparin increases plasmin activity,24 which directly degrades extracellular matrix components such as laminin and fibronectin,25 resulting in impaired vascular integrity and induction of hemorrhage. Dietrich et al26 reported that the photochemical approach accentuates downstream blood-brain barrier dysfunction; this is also a likely exacerbative factor that contributes to hemorrhage.
Some studies have shown that free radical scavengers such as the spin-trap agents and others reduce infarct volume after ischemia or ischemia-reperfusion; although the improvements in neurological scores were not reported,27,28 
-phenyl-tert-butyl-nitrone and oxypurinol were reported to be effective in improving neurological scores.29,30 In the previous study, we demonstrated neuroprotective effects of EPC-K1 in focal cerebral ischemia in rats.31 However, in the present study, EPC-K1 reduced infarct volume but did not significantly improve neurological scores. This result may be attributable to mild reduction in infarct volume. This suggests that reduction in infarct volume by the free radical scavenger is unlikely to achieve an optimal protection of brain. EPC-K1 did not prevent hemorrhage in the vehicle-treated group but reduced markedly heparin-produced hemorrhage. Lapchak et al8 also reported that 2 spin-trap agents (N-t-butyl-phenylnitrone and 2,2,6,6-tetramethylpiperidine-N-oxyl) failed to reduce the rate and volume of hemorrhage when administered alone. The reason for hemorrhage in the control rabbits is unclear. Because EPC-K1 is a scavenger of hydroxyl radicals,32 it is possible that this hemorrhage was caused by superoxide radicals or other radicals. Further study should be undertaken to evaluate the mechanism of hemorrhage that occurred naturally after stroke.
In the present study, combination therapy with EPC-K1, which was administered twice, at 3 and 6 hours after ischemia, had a marked effect on reduction in hemorrhage due to heparin, with significantly improved infarct volume and neurological symptoms. The reduction in infarct volume with the combination of EPC-K1 and heparin was significantly greater than that with EPC-K1 alone. The results demonstrate that when the vasculature is pharmacologically protected, the beneficial effects of heparin on reduction in infarct volume are uncovered. This result is consistent with recent studies that demonstrated the spin-trap agents reduced thrombolysis-induced hemorrhage.8,33 These findings suggest that free radicals may be the major mediator in neuronal cell death and that the hydroxyl radical may be the most likely candidate for vascular wall damage.34 The exact molecular mechanisms involved here are not determined; it is possible that EPC-K1, which inhibits hydroxyl radicals, reduces the expression of MMPs and ameliorates the aggravation of basal lamina damage after cerebral ischemia.
In conclusion, the delayed administration of heparin initiated 3 hours after photothrombosis increased intracerebral hemorrhage, which limited its efficacy and resulted in deteriorating neurological symptoms in rabbits. Reduction in heparin-produced hemorrhage and improvement of neurological symptoms by the free radical scavenger suggest that free radical formation may play a key role in intracerebral hemorrhage produced by heparin. This may provide some implications for the treatment of acute stroke in humans.
|
Acknowledgments |
|---|
This work was supported by a Ministry of Education, Science and Culture in Japan grant-in aid for scientific research (12672211).
Received February 14, 2001; revision received June 1, 2001; accepted June 14, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. del Zoppo GJ. Antithrombotic treatments in acute ischemic stroke. Thromb Haemost. 1999; 82: 938–946.[Medline] [Order article via Infotrieve]
2.
Petty GW, Brown RD Jr, Whisnant JP, Sicks JD, O’Fallon WM, Wiebers DO. Frequency of major complications of aspirin, warfarin, and intravenous heparin for secondary stroke prevention. Ann Intern Med. 1999; 130: 14–22.
3. Heo JH, Lucero J, Abumiya T, Koziol JA, Copeland BR, del Zoppo GJ. Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. J Cereb Blood Flow Metab. 1999; 19: 624–633.[Medline] [Order article via Infotrieve]
4. Gasche Y, Fujimura M, Morita-Fujimura Y, Copin JC, Kawase M, Massengale J, Chan PH. Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: a possible role in blood-brain barrier dysfunction. J Cereb Blood Flow Metab. 1999; 19: 1020–1028.[Medline] [Order article via Infotrieve]
5.
Rosenberg GA, Navratil M. Metalloproteinase inhibition blocks edema in intracerebral hemorrhage in the rat. Neurology. 1997; 48: 921–926.
6. Morita-Fujimura Y, Fujimura M, Gasche Y, Copin JC, Chan PH. Overexpression of copper and zinc superoxide dismutase in transgenic mice prevents the induction and activation of matrix metalloproteinases after cold injury-induced brain trauma. J Cereb Blood Flow Metab. 2000; 20: 130–138.[Medline] [Order article via Infotrieve]
7.
Lapchak PA, Chapman DF, Zivin JA. Metalloproteinase inhibition reduces thrombolytic (tissue plasminogen activator)-induced hemorrhage after thromboembolic stroke. Stroke. 2000; 31: 3034–3040.
8.
Lapchak PA, Chapman DF, Zivin JA. Pharmacological effects of the spin trap agents N-t-butyl-phenylnitrone (PBN) and 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) in a rabbit thromboembolic stroke model: combination studies with the thrombolytic tissue plasminogen activator. Stroke. 2001; 32: 147–153.
9. Zhao B-Q, Suzuki Y, Kondo K, Kawano K, Ikeda Y, Umemura K. Cerebral hemorrhage due to heparin limits its neuroprotective effects: studies in a rabbit model of photothrombotic middle cerebral artery occlusion. Brain Res. 2001; 902: 30–39.[Medline] [Order article via Infotrieve]
10. Kawano K, Fujishima K, Ikeda Y, Kondo K, Umemura K. ME3277, a GPIIb/IIIa antagonist, reduces cerebral infarction without enhancing intracranial hemorrhage in photothrombotic occlusion of rabbit middle cerebral artery. J Cereb Blood Flow Metab. 2000; 20: 988–997.[Medline] [Order article via Infotrieve]
11. Kawano K, Ikeda Y, Kondo K, Umemura K. Increased cerebral infarction by cyclic flow reductions: studies in the guinea pig MCA thrombosis model. Am J Physiol. 1998; 275: R1578–R1583.
12.
Slivka A, Pulsinelli W. Hemorrhagic complications of thrombolytic therapy in experimental stroke. Stroke. 1987; 18: 1148–1156.
13. Wei T, Chen C, Li F, Zhao B, Hou J, Xin W, Mori A. Antioxidant properties of EPC-K1: a study on mechanisms. Biophys Chem. 1999; 77: 153–160.[Medline] [Order article via Infotrieve]
14.
Umemura K, Wada K, Uematsu T, Nakashima M. Evaluation of the combination of a tissue-type plasminogen activator, SUN9216, and a thromboxane A2 receptor antagonist, vapiprost, in a rat middle cerebral artery thrombosis model. Stroke. 1993; 24: 1077–1081.
15. Watson BD, Dietrich WD, Prado R, Ginsberg MD. Argon laser-induced arterial photothrombosis: characterization and possible application to therapy of arteriovenous malformations. J Neurosurg. 1987; 66: 748–754.[Medline] [Order article via Infotrieve]
16.
Yao H, Ginsberg MD, Watson BD, Prado R, Dietrich WD, Kraydieh S, Busto R. Failure of MK-801 to reduce infarct volume in thrombotic middle cerebral artery occlusion in rats. Stroke. 1993; 24: 864–870.
17. Saniabadi AR, Umemura K, Matsumoto N, Sakuma S, Nakashima M. Vessel wall injury and arterial thrombosis induced by a photochemical reaction. Thromb Haemost. 1995; 73: 868–872.[Medline] [Order article via Infotrieve]
18. Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD. Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol. 1985; 17: 497–504.[Medline] [Order article via Infotrieve]
19. Umemura K, Wada K, Uematsu T, Mizuno A, Nakashima M. Effect of 21-aminosteroid lipid peroxidation inhibitor, U74006F, in the rat middle cerebral artery occlusion model. Eur J Pharmacol. 1994; 251: 69–74.[Medline] [Order article via Infotrieve]
20. Alexandrov AV, Bladin CF, Norris JW. Intracranial blood flow velocities in acute ischemic stroke. Stroke. 1994; 25: 1378–1383.[Abstract]
21. Klaassen CD. Pharmacokinetics of rose bengal in the rat, rabbit, dog and guinea pig. Toxicol Appl Pharmacol. 1976; 38: 85–100.[Medline] [Order article via Infotrieve]
22. Majerus PW, Broze GJ Jr, Miletich JP, Tollefsen DM. Anticoagulant, thrombolytic, and antiplatelet drugs.In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG, eds. Goodman and Gilman’s The Pharmacological Basis of Therapeutics. New York, NY: McGraw-Hill; 1996: 1343–1346.
23. Overgaard K, Pedersen H, Knudsen JB, Boysen G. Fatal ischemic brain edema after unsuccessful thrombolysis. Neurol Res. 1990; 12: 81–82.[Medline] [Order article via Infotrieve]
24. Fareed J, Walenga JM, Hoppensteadt DA, Messmore HL. Studies on the profibrinolytic actions of heparin and its fractions. Semin Thromb Hemost. 1985; 11: 199–207.[Medline] [Order article via Infotrieve]
25.
Liotta LA, Goldfarb RH, Brundage R, Siegal GP, Terranova V, Garbisa S. Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane. Cancer Res. 1981; 41: 4629–4636.
26. Dietrich WD, Prado R, Watson BD, Nakayama H. Middle cerebral artery thrombosis: acute blood-brain barrier consequences. J Neuropathol Exp Neurol. 1988; 47: 443–451.[Medline] [Order article via Infotrieve]
27. Takasago T, Peters EE, Graham DI, Masayasu H, Macrae IM. Neuroprotective efficacy of ebselen, an anti-oxidant with anti-inflammatory actions, in a rodent model of permanent middle cerebral artery occlusion. Br J Pharmacol. 1997; 122: 1251–1256.[Medline] [Order article via Infotrieve]
28. Lancelot E, Callebert J, Revaud ML, Boulu RG, Plotkine M. Detection of hydroxyl radicals in rat striatum during transient focal cerebral ischemia: possible implication in tissue damage. Neurosci Lett. 1995; 197: 85–88.[Medline] [Order article via Infotrieve]
29. Cao X, Phillis JW. Alpha-phenyl-tert-butyl-nitrone reduces cortical infarct and edema in rats subjected to focal ischemia. Brain Res. 1994; 644: 267–272.[Medline] [Order article via Infotrieve]
30. Lin Y, Phillis JW. Deoxycoformycin and oxypurinol: protection against focal ischemic brain injury in the rat. Brain Res. 1992; 571: 272–280.[Medline] [Order article via Infotrieve]
31. Takamatsu H, Kondo K, Ikeda Y, Umemura K. Hydroxyl radical generation after the third hour following ischemia contributes to brain damage. Eur J Pharmacol. 1998; 352: 165–169.[Medline] [Order article via Infotrieve]
32. Mori A, Wdamatsu R, Kohno M, Ohmori S. A new hydroxyl radical scavenger: EPC-K1. Neurosciences. 1989; 15: 371–376.
33. Asahi M, Asahi K, Wang X, Lo EH. Reduction of tissue plasminogen activator-induced hemorrhage and brain injury by free radical spin trapping after embolic focal cerebral ischemia in rats. J Cereb Blood Flow Metab. 2000; 20: 452–457.[Medline] [Order article via Infotrieve]
34.
Kontos HA, George E. Brown Memorial Lecture. Oxygen radicals in cerebral vascular injury. Circ Res. 1985; 57: 508–516.
This article has been cited by other articles:
 |
|
 |
|
  M. Fisher, A. Davalos, A. Rogalewski, A. Schneider, E. B. Ringelstein, and W.-R. Schabitz Toward a Multimodal Neuroprotective Treatment of Stroke Stroke, April 1, 2006; 37(4): 1129 - 1136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-Q. Zhao, Y. Ikeda, H. Ihara, T. Urano, W. Fan, S. Mikawa, Y. Suzuki, K. Kondo, K. Sato, N. Nagai, et al. Essential role of endogenous tissue plasminogen activator through matrix metalloproteinase 9 induction and expression on heparin-produced cerebral hemorrhage after cerebral ischemia in mice Blood, April 1, 2004; 103(7): 2610 - 2616. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||