(Stroke. 2001;32:1000.)
© 2001 American Heart Association, Inc.
Original Contributions |
From the Oregon Stroke Center, Department of Neurology, Oregon Health Sciences University, Portland, and the Departments of Neurology and Obstetrics/Gynecology, University of Vermont, Burlington (M.J.C.).
Correspondence to Wayne M. Clark, MD, Oregon Stroke Center, UHS 44, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201. E-mail clarkw{at}ohsu.edu
| Abstract |
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-lipoic acid (LA) are commercially available "antioxidant
supplements" with a variety of actions that may be beneficial during
acute stroke. These actions include inhibiting platelet and
leukocyte activation and adhesion, reducing free radical generation,
and increasing cerebral blood flow. Both EGb and LA have been shown to
be neuroprotective in cell culture and global central nervous system
ischemia models. In this study we investigated the
neuroprotective efficacy of EGb and LA in a clinically relevant,
transient focal central nervous system ischemic
model. MethodsIn the EGb study, 60 adult C57blk mice were randomized to treatment with EGb given orally (via gavage) for 7 days: low dose, 50 EGb mg/kg daily; high dose, 100 mg/kg daily; matched placebo. On day 7, reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 45 minutes followed by reperfusion. At 24 hours, the animals were evaluated on a 28-point clinical scale, and infarct volume was determined with the use of triphenyltetrazolium chloride. In the LA study, 24 C57blk mice were treated with 100 mg/kg SC of LA or placebo 1.5 hours before transient MCAO, as in the EGb study.
ResultsIn the EGb study, values for infarct volume at 24 hours were as follows (mean±SD): low dose (n=18), 13±5 mm3; high dose (n=22), 22±12 mm3; placebo (n=20), 20±10 mm3 (P=0.03 overall; P=0.02, low dose versus placebo). Infarct percentage of hemisphere values were as follows: low dose, 14±5%; high dose, 21±11%; placebo, 20±9% (P=0.03 overall; P=0.02, low dose versus placebo). Ten percent of the high-dose group showed significant intracerebral hemorrhage (ICH) within the infarct, while no ICH was seen in the other groups. Neurological function scores were as follows: low dose, 11.8±1.5; high dose, 11.4±1.7; placebo, 11.3±1.8 (P=NS). In the LA study, infarct volume was as follows: 100 mg/kg LA (n=12), 16.8±8.3 mm3; placebo (n=12), 27.2±14.6 mm3 (P<0.05). LA also produced a significant improvement in neurological function at 24 hours: LA, 9.5±1.2; placebo, 11.2±1.8 (P=0.02). There was no evidence of ICH in any of the animals.
ConclusionsBoth oral EGb and LA therapies produced significant reductions in stroke infarct volume. However, for EGb this beneficial effect appears to be dose related, with higher doses potentially increasing the risk of ICH.
Key Words: animal models antioxidants neuroprotection oxygen radical mice
| Introduction |
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-lipoic acid (LA), have a
multitude of biological effects that potentially may reduce reperfusion
injury.4 EGb is extracted from the leaves and nuts of the Ginkgo biloba tree and has been used for centuries in Asia and Europe for a variety of disorders.4 It has recently been recognized in the United States after receiving Food and Drug Administration approval for clinical study in memory disorders.4 5 EGb extract contains flavonoids, which appear to possess strong free radical scavenging and anti-inflammatory properties,5 6 and terpenoids (ginkgolides A and B),7 which inhibit PAF and decrease free radical release.8 Treatment with EGb has shown beneficial effects in reducing reperfusion injury in CNS global ischemia and trauma experimental models.9
LA is a thiol antioxidant (similar to glutathione) that is
absorbed from the diet and crosses the blood-brain
barrier.10 LA is taken up
and reduced in cells to dihydrolipoate (DHLA), which is exported
extracellularly.10 LA and
DHLA have been shown to inhibit nuclear factor-
(NF-
) activation
and decrease leukocyte adhesion receptor
expression.11 LA has been
shown to reduce infarct volume in permanent (clipped) focal middle
cerebral artery occlusion (MCAO) models in both rats and
mice.12 13 Since
the inflammatory response appears to be more important in transient
ischemia (reperfusion injury), LA may be more effective in a
reversible MCAO model.
The overall objective of this study was to investigate the neuroprotective efficacy of EGb and LA in the treatment of CNS ischemic injury with an animal model that closely approximates clinical stroke with reperfusion.
| Materials and Methods |
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CNS Ischemia Model
Transient ischemia was produced by filament
occlusion of the right MCA following a modification of the method
originally reported by Koizumi et
al,14 using nylon
monofilaments (8-0) coated with silicone. The animals were
anesthetized via inhalation mask with 1.25% to 1.5%
halothane/oxygen. A skin incision in the right temporoparietal
area was made, the temporalis muscle was retracted, and a microtip of
the laser-Doppler probe (Periflux System 5000) was glued to the
skull with cyanoacrylate adhesive (Krazy Glue). Continuous cerebral
blood flow was recorded throughout the experiment.
A rectal temperature probe was introduced, and a heating pad maintained body temperature at 37±0.5°C. Under a dissecting microscope, the right carotid bifurcation was exposed, and the external carotid artery was coagulated distal to the bifurcation. After temporary ligature of the common carotid artery, an 8-0 coated nylon filament was inserted through the external carotid artery stump and gently advanced (8 to 9 mm) to occlude the origin of the MCA. Successful MCAO was confirmed by laser Doppler, with a criterion of <20% of baseline blood flow remaining after filament placement. For the following 45 minutes the animal remained untouched under constant conditions. At the end of the period, the filament was withdrawn, the external carotid artery stump was tied, the common carotid artery was untied, and reperfusion was confirmed by laser Doppler (>85% of baseline). The animals were then permitted to recover from anesthesia on a warming pad (at 24°C) and sheltered from drafts.
Neurological Function and Infarct Size
Determination
At 24 hours the animals were scored for neurological
deficit by a 28-point focal scoring system that we have
developed.15 Two observers,
blinded to groups, scored the animals independently, and the scores
were averaged. The animals were then anesthetized
(intraperitoneal
ketamine/xylazine/acepromazine), and the brain was quickly
removed and embedded in 1.5% agarose and sectioned with a Stoelting
tissue slicer into six 1-mm slices. These slices were placed in a well
plate containing 2% tetrazolium chloride solution and allowed to
develop for 30 minutes. The tissue was then placed in 10% formalin and
scanned, and images were placed in Adobe Photoshop. The National
Institutes of Health Image Analysis program was then used to
measure the area of ischemia and total hemisphere area. Volume
was calculated by multiplying the 1-mm slice thickness of the measured
areas. To partially correct for effects of edema, all lesions were also
expressed as percentage of ipsilateral hemisphere. Two investigators
independently imaged the sections.
All results are expressed as mean±SD. ANOVA (EGb study) and unpaired Students t test (Bonferroni corrected) were used to assess the significance of differences in lesion volumes (P<0.05).
| Results |
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LA Study
A total of 24 animals were evaluated. No side effects
were seen, and no differences in weight or cerebral blood flow were
seen. Treatment with LA produced a significant reduction in lesion size
at 24 hours: 100 mg/kg LA (n=12), 16.8±8.3
mm3; placebo (n=12), 27.2±14.6
(P<0.05;
df=21;
t test, 2.08). LA also produced
a significant improvement in neurological function at 24 hours: LA,
9.5±1.2; placebo, 11.2±1.8
(P=0.02;
df=21;
t test, 2.49). There was no
evidence of ICH in any of the animals.
| Discussion |
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This study did not investigate whether EGb and LA would still be effective if given after the onset of ischemia; this is planned in future studies. In addition, all animals in the present study were evaluated at 24 hours. As such, these results represent early ischemic injury events, including cerebral edema, and do not address whether these agents have an effect in later recovery from stroke. Further study involving evaluations at later time periods are needed to answer this question.
In this study we found a 35% to 40% relative reduction in lesion size at 24 hours with EGb and LA. The magnitude of this reduction is similar to that seen in other studies involving antireperfusion injury agents in this model.16 17 18 19 20 We saw a treatment effect on functional outcome at 24 hours with LA but not EGb in this study. As is the case in clinical stroke, infarct size in the MCAO model does not always correlate with outcome. In prior studies we found a correlation of r=0.76 (R2=0.49) between the 28-point examination and infarct volume. It is unclear why a corresponding neurological improvement was not seen with EGb treatment. This study also does not allow for a direct comparison of the relative efficacy or safety of EGb and LA because different treatment paradigms were used for the 2 agents.
Although the exact neuroprotective mechanism of EGb is not known, there is strong support for its role as a putative antireperfusion injury therapy. The flavonoid components have been shown to be responsible for directly scavenging free radicals, decreasing lipid peroxidation, and reducing free radical generation in experimental ischemia/reperfusion models.21 22 The terpenoid component (ginkgolides A and B) of EGb has been shown to improve blood flow and reduce thrombus formation by inhibiting PAF.23 24 The ginkgolides have also been shown to inhibit neutrophil activation, adhesion, and infiltration during reperfusion.25 26 EGb has also been shown to be beneficial in experimental global CNS ischemia models. Preischemic administration of ginkgolide B (50 mg/kg) was found to significantly reduce neuronal necrosis at 7 days in a transient forebrain ischemia model in rats.27 In the same model, Krieglstein et al28 found that EGb (100/kg IV) given 45 minutes after ischemia was able to increase cerebral blood flow and diminish delayed hypoperfusion. EGb also produced beneficial effects on functional outcome in a cerebral microemboli model in rats29 and in a global cerebral ischemia model in dogs.30 Clinically, EGb is readily available and widely used for the treatment of memory disorders. Numerous trials have found small but significant beneficial effects of EGb (120 to 240 mg/d) on memory and social function in early dementia.31 32 33 34 Potential bleeding complications of EGb are a concern since this population is also at increased risk for ICH because of coexisting hypertension and cerebral amyloid angiopathy. A recent case report described bilateral hematoma formation presumably due to EGb.35 Our study suggests that higher doses of EGb may increase the risk of hemorrhagic transformation after stroke. It is not known whether the risk of ICH would be further increased in patients on concurrent antiplatelet, anticoagulant, or thrombolytic agents. Further dose finding, along with combination agent experimental stroke studies, may help to answer these questions.
The mechanism of action of LA appears to be due to its
ability to substitute for glutathione. One of the major consequences of
free radical injury is the depletion of the cellular antioxidant
glutathione, leading to oxidation of protein thiols to disulfides and
the loss of enzymes having thiol groups. Glutathione itself cannot be
administered directly; however, LA is a thiol antioxidant that is
absorbed from the diet and crosses the blood-brain
barrier.10 Four antioxidant
properties have been demonstrated: it has metal chelating capacity; it
can scavenge reactive oxygen species; it can regenerate
endogenous antioxidants, including vitamins E and C; and it
can repair oxidative
damage.10 LA and DHLA have
been shown to inhibit NF-
activation and decrease adhesion receptor
expression.36 LA has been
shown to be neuroprotective in several studies involving focal CNS
ischemia. Wolz and
Krieglstein12 studied LA in
permanent focal MCAO models in both rats and mice when infarct volume
was evaluated at 48 hours. In rats, 100 mg/kg SC of LA injected 2 hours
before MCAO produced a significant reduction in infarct volume. In
mice, a dose of 100 mg/kg SC at either 1 or 2 hours before
ischemia produced a significant reduction in volume. Using the
same permanent MCAO model, Prehn et
al13 found that a dose of 50
mg/kg SC of DHLA given 30 minutes before ischemia produced
significant infarct reduction in rats and mice. Panigrahi et
al37 found that pretreatment
with 25 mg of LA just before reversible global ischemia
significantly improved mortality from 79% to 25%. Finally, Mitsui et
al38 found that 100 mg/kg of
LA given before ischemia produced significant protection in a
peripheral nerve ischemia/reperfusion model. All of
these successful studies, including ours, used pretreatment strategies.
Although this might be clinically relevant if a patient was taking LA
supplements before stroke, it does not address the potential of LA as
an acute poststroke therapy. However, it appears that LA was well
tolerated in all these trials, without any reports of ICH.
Conclusion
This study found that pretreatment with 2 commonly used
antioxidant supplements, EGb and LA, reduced neurological injury in a
transient focal CNS ischemic model. For EGb this protective
effect appears to be dose sensitive, with higher dose of EGb
potentially increasing the risk of ICH. The combined clinical use of
EGb with antiplatelet, anticoagulant, and
thrombolytic agents could potentially further increase
the risk of ICH. Additional studies investigating the efficacy of
delayed treatment and safety of these supplements in experimental
stroke are needed before clinical studies are
undertaken.
| Acknowledgments |
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Received October 4, 2000; revision received December 1, 2000; accepted December 13, 2000.
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