(Stroke. 1997;28:2018-2024.)
© 1997 American Heart Association, Inc.
Articles |
Superior Neuroprotective Efficacy of a Novel Antioxidant (U-101033E) With Improved Blood-Brain Barrier Permeability in Focal Cerebral Ischemia
From the Department of Neurosurgery (R.S.-E., S.Z., H.-J.R) and Institute for Surgical Research (E.H., N.P., A.B.), Klinikum Großhadern, Ludwig-Maximilians-University, Munich, Germany.
Correspondence to Dr Robert Schmid-Elsaesser, Department of Neurosurgery, Ludwig Maximilians University, Klinikum Grosshadern, Marchioninistr 15, 81377 Munich, Germany.
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Abstract |
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Background and Purpose The vascular endothelium and parenchyma of the brain have the potential to generate free radicals under pathological conditions, but it is unclear which of these two sites prevails in the production of free radicals and should be the primary target of therapeutic intervention. To clarify this issue, we compared the neuroprotective properties of a 21-aminosteroid (U-74389G) that acts on the microvasculature and a pyrrolopyrimidine (U-101033E), a novel antioxidant compound that has significantly improved potential to enter the brain parenchyma.
Methods In Sprague-Dawley rats the middle cerebral artery was occluded for 90 minutes by an intraluminal filament. Local cortical blood flow was recorded by bilateral laser Doppler flowmetry throughout ischemia and 1 hour of reperfusion. Three groups of rats were studied: controls that received vehicle only and animals that received either U-74389G or U-101033E. Neurological examinations were performed daily, and infarct size was assessed histologically 7 days after ischemia.
Results U-101033E reduced infarct volume significantly by 51%, whereas U-74389G led to a nonsignificant decrease in infarct volume. U-101033E improved neurological function immediately after ischemia, whereas U-74389G led to improvement only at the end of the observation period. Laser Doppler measurements showed no significant difference in local cortical blood flow among the treatment groups.
Conclusions We conclude that for treatment of transient focal ischemia, an antioxidant that crosses the blood-brain barrier might be superior to agents that predominantly act on the endothelium of the cerebral microvasculature.
Key Words: cerebral ischemia, focal • free radicals • lipid peroxidation • neuroprotection • rats
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Introduction |
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Oxygen free radicals have been postulated to play a critical role in acute postischemic neuronal degeneration.1 2 Thus, efforts have been undertaken to develop antioxidants that attenuate the secondary neuronal injury. The 21-aminosteroids (lazaroids) are potent inhibitors of free radical–induced lipid peroxidation,3 4 and they have multiple antioxidant actions: (1) scavenging of lipid peroxyl; (2) decreasing formation or scavenging of hydroxyl radicals; (3) reducing lipid peroxidation–induced arachidonic acid release; (4) maintaining the levels of endogenous vitamin E; and (5) membrane stabilization by decreasing membrane fluidity.5 The 21-aminosteroid tirilazad mesylate (U-74006F) is currently being investigated in phase III clinical trials in patients with head or spinal cord injury, subarachnoid hemorrhage, or ischemic stroke.6 U-74389G differs structurally from tirilazad mesylate (U-74006F) only by the absence of the methyl group in the 16-position of the steroid ring system (Fig 1
), and the two drugs have similar
pharmacodynamic and pharma- cokinetic profiles.4 Because both drugs are
lipophilic compounds that accumulate in cell membranes with a high
affinity for vascular endothelium, they may protect the
vascular endothelium from peroxidative damage but are
not expected to cross the BBB.7
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Recently discovered antioxidants, the pyrrolopyrimidines, have a
significantly greater potential to enter the brain
parenchyma.8 Physiochemical studies4 7
indicate that the highly lipophilic steroid moiety of the
21-aminosteroids orients itself within the hydrophobic fatty acid core
of the membrane and is largely responsible for the high affinity for
cell membranes.8 The pyrrolopyrimidine U-101033E lacks the
lipophilic steroid moiety present on the lazaroids (Fig 1
), which
reduces the affinity of pyrrolopyrimidine for vascular
endothelium and tendency to be retained in lipid
bilayers (Raub and Sawada, unpublished data in Hall et
al8 ). Within 5 minutes of intravenous
administration, a significantly greater concentration of
pyrrolopyrimidines is found in brain tissue compared with tirilazad.
However, the pyrrolopyrimidine diffuses out of the brain more quickly
after each dose.8
It has been proposed that free radical injury of the brain mainly involves the microvasculature,9 10 11 although both brain parenchyma and vascular endothelium have the potential to generate free radicals.1 12 Because it is unclear which of these compartments predominates in the production of free radicals13 and should be the primary target of therapeutic interventions, we compared the neuroprotective properties of the 21-aminosteroid U-74389G, which primarily acts on the microvasculature, and the pyrrolopyrimidine U-101033E, which is able to cross the BBB. Preliminary data were presented as an abstract.14
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Materials and Methods |
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Thirty-two male Sprague-Dawley rats weighing 250 to 300 g were used in this study. Animals were purchased from Charles River Laboratory (Sulzfeld, Germany). All procedures involving the animals were conducted according to institutional guidelines and were in compliance with regulations formulated by the government of Bavaria.
All animals were fasted overnight before the experiment, administered atropine (0.5 mg/kg) subcutaneously, and anesthetized with 4% halothane. The rats were then intubated orotracheally and mechanically ventilated with 0.8% halothane in a mixture of 70% N2O and 30% O2 to maintain normal arterial blood gases. Temporalis muscle and rectal probes were used to monitor temperature throughout the experiment, and a thermostatically regulated heating lamp and pad were used to maintain temperature at 37°C. The tail artery and left femoral vein were cannulated for blood sampling, monitoring of arterial blood pressure, and administration of fluids and drugs. Serum glucose was measured before ischemia. Arterial blood gases, hemoglobin, and hematocrit were measured before, during, and after ischemia. Local cortical blood flow was monitored in the cerebral cortex of each hemisphere in the supply territory of the middle cerebral artery by laser Doppler flowmetry (MBF3D, Moor Instruments Ltd).
Bilateral burr holes (1 mm in diameter) were drilled 5 mm lateral and 1 mm posterior to the bregma without injury to the dura mater. Then each animal was placed supine, and the head was firmly immobilized in a stereotaxic frame. A rectangularly bent laser Doppler probe was positioned over each brain hemisphere for continuous measurement of local cortical blood flow (2-Hz sampling rate) from before the onset of ischemia until 1 hour after reperfusion. Flow values were calculated as averaged values during 1-minute periods every 10 minutes, with shorter intervals immediately after induction of ischemia and reperfusion.
All rats were subjected to 90 minutes of right MCAO by the introduction of a silicone-coated 4-0 nylon monofilament via the external carotid artery, with the use of the procedure described by Koizumi et al.15 Intra-arterial injections of heparin (150 IU/kg) were administered before induction of ischemia and 1 hour afterward to prevent blood clotting. Animals in which laser Doppler flowmetry showed a marked decrease in contralateral flow shortly after insertion of the filament, indicating subarachnoid hemorrhage,16 17 were excluded.
The rats were randomly assigned to one of three treatment arms (n=10 each) that received isovolumetric doses of (1) vehicle, (2) U-74389G, or (3) U-101033E. Drugs were administered 15 minutes before onset of ischemia, 90 minutes later during ischemia, and 60 minutes after reperfusion. Each dose consisted of 3.0 mg/kg dissolved in 0.02 mol/L citric acid to equal a concentration of 1 mg/mL and was administered intravenously over 15 minutes.
Neurological function was evaluated daily by a "blinded" coworker using a grading scale of 0 (no spontaneous activity) to 5 (no apparent deficit) that was modified from that described by Bederson et al.18 The rectal temperature was measured daily during the observation period to rule out postischemic hyperthermia.19 20 Seven days after ischemia, the rats were anesthetized by chloral hydrate and perfused transcardially by isotonic heparinized saline, followed by 2% paraformaldehyde for fixation of tissues. The brains were removed, embedded in paraffin, and cut in the coronal plane into 4-µm-thick sections at 400-µm intervals. The brain slices were stained with hematoxylin and eosin, and the infarct areas were assessed planimetrically (OPTIMAS 5.1, BioScan Inc) by a blinded examiner. For each brain, 24 slices were measured containing the entire infarct. The total infarct volume was calculated by multiplying the total infarct area of each slice by the distance (400 µm) between successive slices and was expressed as percentage of the contralateral hemisphere. Infarct volumes of the cortex and basal ganglia were determined by measuring the area of infarct in sections obtained 2, 3.6, 5.2, 6.8, and 8.4 mm from the frontal pole. Cortex and basal ganglia were determined according to a stereotaxic atlas of the rat brain.21 The volumes of infarcts were expressed as percentages of the volumes of the contralateral cortex or basal ganglia. Averages were calculated for the total infarct volume and volumes of cortical and basal ganglia infarcts in each group, and these values were used for comparison.
Statistical analysis was performed with the use of SigmaStat 2.0 Statistical Software (Jandel Scientific). Physiological data at each time point and infarct volumes were analyzed with one-way ANOVA, laser Doppler data with two-way ANOVA for repeated measures, and neurological function scores with Kruskal-Wallis ANOVA on ranks for each of 7 days. If multiple comparisons were indicated, the nonparametric Dunnett's test for neurological function scores was applied. Significance was accepted at the P<.05 level. Results are presented as mean±SD.
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Results |
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Two rats had indications of subarachnoid hemorrhage after insertion of the occluding filament and were excluded from the study. The results of physiological monitoring of the remaining rats are presented in the Table
. The
experimental groups did not differ with respect to
preischemic, intraischemic, or
postischemic blood pressure or arterial blood
gases. There were no differences in hemoglobin, hematocrit, or serum
glucose between the groups. All animals remained
normothermic during surgery and the 7-day postoperative
observation period. Postischemic hyperthermia, which was
reported to occur in the intraluminal thread model of cerebral
ischemia,19 20 was not observed in our study.
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Laser Doppler flow measurements showed that MCAO resulted in a
significant reduction of microcirculatory blood flow to 20% to 30% of
baseline in the ipsilateral tissue area, ie, in the core region of
infarction, in all groups, which is consistent with findings in
other studies.22 23 24 After reperfusion,
postischemic hyperemia was followed by a gradual
decrease in blood flow to approximately 70% of baseline. Delayed
hypoperfusion persisted until the end of the recording period.
Cortical blood flow in the contralateral hemisphere remained unchanged
throughout the experiment. There was no difference in local cortical
blood flow between treatment groups and the control group (Fig 2).
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Treatment with U-101033E improved neurological function immediately
after ischemia, whereas animals treated with U-74389G showed
better neurological function than controls only at the end of the
experimental observation period (Fig 3).
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The total volume of infarction was 20.5±10.5% in controls,
15.5±11.2% in animals that received U-74389G, and
10.0±7.2% in animals that received U-101033E (mean±SD,
percentage of contralateral hemisphere) (Fig 4). U-101033E significantly
(P<.05) reduced total infarct volume by 51% compared with
controls, whereas U-74389G led to a nonsignificant 24% reduction.
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Both agents were found to protect cortical brain tissue. U-101033E
limited infarction in the cortex to 9.8±9.2% and U-74389G limited
infarction to 16.0±15.0% compared with a cortical infarct volume of
26.1±20.5% in untreated controls (mean±SD, percentage of
contralateral cortex volume) (Fig 5).
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Only U-101033E was significantly effective in protecting the basal
ganglia from infarction. The volume of infarction in the basal ganglia
of animals receiving U-101033E was restricted to 7.6±5.8% compared
with an infarction volume of 16.6±6.5% in animals receiving vehicle
only (mean±SD, percentage of contralateral basal ganglia volume). The
limitation of infarction to 12.9±8.8% of contralateral basal ganglia
volume in U-74389G–treated rats did not reach a statistically
significant level (Fig 5).
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Discussion |
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As demonstrated in this study, U-101033E is more effective than the 21-aminosteroid in protecting neuronal tissue from the effects of focal cerebral ischemia because it better preserved neurological function, reduced total infarct volume by 51%, and protected the basal ganglia. This may indicate that antioxidants, which are able to cross the BBB, may have a greater potential to treat cerebral ischemia.
The marked difference in efficacy in this study between the 21-aminosteroid and pyrrolopyrimidine raises the question of where in the central nervous system radicals are generated and where they act. Both parenchyma and the vascular endothelium possess potential pathways for the production of radicals,1 9 10 12 but it is unclear in which of these compartments the main production of radicals takes place.13
The high density of mitochondria and concentration of polyunsaturated fatty acids and xanthine oxidase in the cerebral endothelium suggest that this tissue is involved in the generation of oxidative radicals.25 In fact, Grammas et al9 have shown that isolated brain microvessels subjected to anoxia produce large amounts of hydroxyl radicals upon reoxygenation. Kumar et al10 have demonstrated that cerebral microvessel endothelial cells in culture, in the absence of neutrophils, produce hydroxyl radicals and nitric oxide, confirming that the brain vasculature is a source of reactive oxygen formation.
The damage caused by hydroxyl radicals is spatially highly restricted. This is due to the molecule's short half-life of only a few microseconds, which limits its diffusion from the site of production to 10 nm or less.1 26 Other radicals or radical precursors may diffuse from the site of formation and cause damage at distant loci. Kontos et al27 have provided evidence that in fluid percussion brain injury or acute hypertension, oxygen radicals are discharged into the extracellular space through anion channels. Thus, superoxide may appear in the extracellular space in close proximity to the cerebral blood vessels. The finding that the appearance of superoxide can be inhibited by anion channel blockers suggests that these radicals are derived from endothelial cells.27 28
Lipid peroxide is another important radical species. Enzymatic oxidation of arachidonic acid produces reactive oxygen, which initiates lipid peroxidation. In hypertensive cerebrovascular injury, oxidation of arachidonate was found to occur selectively in cerebral arterioles and not in the brain parenchyma.29 In summary, assuming that the microvasculature is an important locus of free radical formation, and given the fact that radical-induced injury most frequently occurs in structures of the vasculature proper, it can be expected that administration of endothelial-targeted antioxidants would interrupt lipid peroxidation initiated by oxygen radicals and that this would offer benefits for the treatment of cerebral ischemia.11 30
On the other hand, neuronal injury can also be induced by oxygen radicals produced in brain parenchyma itself, ie, far away from the endothelium. Evidence for the production of radicals in brain has predominantly been provided by studies of brain tissue homogenates and mitochondrial preparations in which the contributions from endothelium are negligible.13 Microdialysis studies of local hydroxyl radical production have also shown that the parenchyma produces free radicals when subjected to focal or global ischemia or experimental head trauma.31 The data indicate that reactive oxygen species generated by mitochondrial electron transport escape cellular antioxidant defenses and promote highly damaging hydroxyl radical activity after transient cerebral ischemia.32
Literature on the therapeutic efficacy of oxygen radical scavengers suggests that drugs that are able to cross the BBB have the greatest effectiveness in protecting the brain from ischemic damage.33 These agents may be more efficacious because they are lipophilic and thus localize to neuronal membranes, where they can more effectively inhibit lipid peroxidation.13 Furthermore, brain-penetrating antioxidants could accumulate in the mitochondria, where they conceivably could prevent free radical damage to components of the respiratory chain.34 If access to intracellular compartments is required for therapeutic efficacy, it appears unlikely that free SOD, a large, water-soluble molecule that cannot readily cross cell membranes, would provide a protective effect. Indeed, treatment with copper-zinc SOD was not effective after global ischemia.35 36 37 Highly potent antioxidants like ascorbate and trolox, an aqueous soluble analogue of vitamin E, failed to show a neuroprotective effect in an MCAO model of transient focal ischemia.38
Antioxidant therapy seems to be more effective when reperfusion of the brain is a major contributor to neuropathology, as shown in studies in transgenic mice, which overexpress human copper-zinc SOD and which proved highly resistant to reperfusion injury associated with transient MCAO.39 40 41 In transient ischemia, reperfusion can be expected to improve the concentration of the drug in postischemic brain, whereas in models with permanent ischemia the drug may only be delivered to tissues supplied by good collateral circulation unless the drug is able to penetrate the tissue to reach the ischemic region. Hall et al8 reported that U-101033E reduced infarct volume by 27% in a mouse model of permanent MCAO, whereas tirilazad was minimally effective in this model. The authors concluded that U-101033E and other pyrrolopyrimidines significantly decreased infarct size, most likely because they were better able to gain access to the ischemic brain parenchyma. In addition, they demonstrated that U-101033E has a greater ability to protect the CA1 region in gerbils after 5 minutes of transient forebrain ischemia and that the therapeutic window is at least 4 hours. Interestingly, tirilazad and certain pyrrolopyrimidines (U-87663E and U-89843A) were equally effective in protecting the hippocampal CA1 region after 3 hours of unilateral carotid artery occlusion and 12 hours of reperfusion in gerbils.8 To explain these findings, the authors speculated that (1) postischemic BBB reperfusion damage might be so severe after 3 hours of ischemia that tirilazad gains access to the parenchyma, or (2) BBB damage and its potential attenuation by microvascularly localized tirilazad are counterbalanced by parenchymal neuronal injury mechanisms, which would be most effectively countered by the brain-penetrating pyrrolopyrimidines.
Other brain-penetrating antioxidants such as liposome-entrapped SOD, allopurinol, and dimethylthiourea have also been shown to be neuroprotective in animal models of permanent focal ischemia42 43 44 45 in which antioxidants localized to the vasculature were ineffective or only minimally effective.46 47 48 Whereas most free radical scavengers reduced infarct size only moderately (by 25% to 35%) after focal ischemia,1 42 49 50 convincing results were obtained with the spin-trapping agent PBN.34 51 52 Nitrone-based spin traps penetrate the brain readily at high concentrations53 and react with transient free radicals to form stable spin adducts.54 55 The reported 50% protection in focal reversible ischemia in rats pretreated with PBN51 lies within the same range as our results with U-101033E. Furthermore, administration of PBN prevented infarction in the densely ischemic lateral portion of the caudoputamen, which is normally infarcted after 30 minutes of MCAO.56 These results were similar to the findings in the present study that U-101033E markedly limited the extent of infarction in the basal ganglia. In another study, PBN-treated animals that had undergone 2 hours of MCAO and 4 hours of reperfusion showed pronounced recovery of energy state with concentrations of ATP and lactate in both focus and penumbra approaching normal values.34 This finding may reflect the partition in cellular organelles or membrane layers57 and emphasizes the importance of the potential therapeutic agent being able to reach the appropriate compartment.
The results of our study indicate that neither U-101033E nor U-74389G alters local cortical blood flow, which leads to the supposition that these antioxidants might exert their beneficial effects by mechanisms other than direct action on cerebral blood flow. On the other hand, we measured local cortical blood flow in the somatosensory cortex near the core of the infarct22 23 during ischemia and through the first hour of reperfusion, and it is possible that improvements in blood flow might have occurred later58 or in more peripheral areas.
Studies of the effects on cerebral blood flow of administering free radical scavengers have had conflicting results.43 48 59 60 61 62 63 In a SOD-1 transgenic mouse model of intraluminal reversible MCAO, Yang et al40 found that reduction of infarct volume and neurological deficits do not depend on changes in cerebral blood flow but rather are correlated with reduced oxidative stress in ischemic brain tissue. The authors propose that the preservation of functional integrity of astroglial cells offers neuronal protection, by mechanisms such as the active reuptake of extracellular glutamate.
We cannot rule out that U-101033 has some unknown neuroprotective mechanisms apart from its antioxidative properties and its ability to cross the BBB. The exact mechanisms by which brain-penetrating antioxidants such as the pyrrolopyrimidines or PBN act remain to be clarified, and this knowledge might provide deeper insight into radical-mediated injury. As our study and studies reported by other authors suggest, antioxidant compounds with greater ability to cross the BBB possibly are better able to protect the brain from the effects of focal ischemia than are antioxidants that predominantly act at the endothelium of the cerebral microvasculature. Treatment with brain-penetrating antioxidants might be more advantageous in cases of focal permanent ischemia, in which microvascular effects seem to be less important, than in cases of temporary ischemia and cases in which the BBB remains intact. Because damage to both the vascular endothelium and brain parenchyma may contribute to radical-mediated injury, and the type of cell that is injured may not be the same type of cell that gives rise to oxygen free radicals, it is conceivable that administering a combination of two or more agents that have antioxidation properties and are able to scavenge free radicals in both blood vessels and brain parenchyma might best protect the brain from free radical–mediated damage.
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Selected Abbreviations and Acronyms |
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-phenyl-N-tert-butyl nitrone
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (Schm1067/2-1) and Friedrich Baur Stiftung. The authors wish to thank Dr Ed Hall of the Upjohn Company, Kalamazoo, Mich, for the generous gift of U-74389G and U-101033E. We also thank Diana Mathis and Dr Urs D. Schmid for editing the manuscript.
Received October 25, 1996; revision received June 27, 1997; accepted July 2, 1997.
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