(Stroke. 1998;29:1002-1006.)
© 1998 American Heart Association, Inc.
Dietary Vitamin E Levels Affect Outcome of Permanent Focal Cerebral Ischemia in Rats
H. B. van der Worp, MD;
P.R. Bär, PhD;
L. J. Kappelle, MD, PhD;
D. J. de Wildt, PharmD, PhD
From the Department of Neurology (H.B. van der W., P.R.B., L.J.K.),
University Hospital Utrecht, and the Department of Medical Pharmacology (D.J.
de W.), Utrecht University, Utrecht, the Netherlands.
Correspondence and reprint requests to H.B. van der Worp, MD, Department of Neurology, University Hospital Utrecht, PO Box 85500, 3508 GA Utrecht, Netherlands. E-mail hworp{at}neuro.azu.nl
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Abstract
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Background and Purpose—A
supraphysiological amount of vitamin E in the
standard diet of laboratory animals may provide partial protection
against cerebral ischemic damage in stroke models. The aim of
the present study was to test the effect of dietary vitamin E on
infarct volume in rats subjected to permanent focal cerebral
ischemia.
Methods—Male Wistar rats were raised on a vitamin E–deficient
diet (n=10) or a control diet containing 62.7 mg vitamin E/kg (n=11)
for 13 to 16 weeks, from the age of 3 weeks. The left middle cerebral
artery (MCA) was permanently occluded by means of an intraluminal
silicone-coated 3–0 suture. Blood flow in the left MCA territory was
measured before and after occlusion with laser Doppler
flowmetry. The area of infarction was measured in
hematoxylin-eosin–stained brain sections by means of an image
analysis system. The investigator was not aware of the vitamin
E status of the rats.
Results—Blood flow in the left MCA territory in the second half
hour after occlusion was 43±17% and 42±17% (mean±SD) of the
baseline value in control and vitamin E–deficient rats, respectively.
The mean infarct volume, measured after 48 hours of survival, was
61±19 mm3 in control rats and 137±76
mm3 in vitamin E–deficient rats
(P=0.037).
Conclusions—After permanent focal cerebral ischemia, the
infarct is larger in vitamin E–deficient rats than in rats raised on a
diet with the usual, supraphysiological amount of
vitamin E. This may have consequences for cerebral ischemia
studies with experimental animals.
Key Words: cerebral ischemia • lipid peroxidation • vitamin E • diet
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Introduction
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Free radical
formation and subsequent lipid peroxidation have been implicated as
important factors in the pathogenesis of ischemic brain
damage.1 2 In animal models, several antioxidants
have been shown to be neuroprotective when given before or shortly
after induction of focal cerebral
ischemia.3 4 5 The naturally occurring
antioxidant vitamin E scavenges lipid peroxyl radicals and thereby
inhibits lipid peroxidation.6 In rats, the extent
of postischemic cerebral lipid peroxidation has been
reported to be dependent on the amount of vitamin E in the
diet.7 To improve health and fertility and to
prevent oxidation of food, animal diets are usually supplemented with
supraphysiological doses of vitamin E. This may
influence outcome in experimental studies of focal cerebral
ischemia.
The aim of the present study was to test the effect of dietary
vitamin E on cerebral infarct volume in rats subjected to permanent
intraluminal middle cerebral artery (MCA) occlusion.
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Materials and Methods
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The experiments were performed according to a protocol approved
by the institutional animal care committee. Male Wistar rats, weaned at
3 weeks of age, were raised on a vitamin E–deficient diet (n=10)
or control diet containing 62.7 mg dl-
-tocopheryl
acetate/kg (n=11) for 13 to 16 weeks. These special diets, containing
5.2% fat, were prepared at our request by Hope Farms (Woerden, the
Netherlands) and differed only in content of
dl--tocopheryl acetate. The composition of the diets was
guaranteed by the manufacturer. Vitamin E concentrations in food
samples from this manufacturer are regularly determined by means of
high-performance liquid chromatography,
according to a method described by the Analytical Methods Committee of
the Royal Society of Chemistry.8 Body weights
(mean±SD) of the rats at onset of the diet were 82±11 and 78±9 g,
respectively. The investigator was blinded to the diet allocation of
the animals during all procedures and measurements.
The rats were anesthetized with 3% isoflurane in a mixture of
70% nitrous oxide and 30% oxygen, intubated, and mechanically
ventilated with a rodent ventilator (Rodent Ventilator 683, Harvard).
Anesthesia was maintained during the operative procedures
and the first 60 minutes after MCA occlusion with 1.5% isoflurane in
the same gas mixture. The left femoral artery was cannulated to
record arterial blood pressure every 30 seconds
(Viggo-Spectramed DT-XX disposable transducer, Viggo-Spectramed BV).
Arterial blood gases were measured before and 30 minutes
after MCA occlusion (model 288 Blood Gas System, Ciba-Corning
Diagnostics Corp). When necessary, respiratory adjustments
were made to maintain normal blood gas values. Rectal temperature was
measured continuously and maintained between 36.5°C and 37.5°C
throughout the experiment by means of a feedback-regulated heating
blanket (homeothermic blanket control unit, Harvard).
MCA occlusion was achieved by a minor modification of the intraluminal
filament technique originally described by Koizumi et
al.9 Briefly, the left common, external,
and internal carotid arteries were identified through a ventral
cervical midline incision. The superior thyroid, maxillary, lingual,
and occipital branches of the external carotid artery were coagulated
and cut. The pterygopalatine artery was ligated with a 5–0 silk
suture. A 3–0 polypropylene monofilament suture, its tip slightly
enlarged by treatment with boiling xylene and coated with silicone, was
introduced into the lumen of the stump of the external carotid artery
and gently advanced into the internal carotid artery (ICA) until a
slight resistance was felt, occluding the origin of the MCA.
To verify MCA occlusion, the regional cerebral blood flow (CBF) in the
cortex supplied by the left MCA was measured every 30 seconds by laser
Doppler flowmetry (LDF) (Periflux PF3, Perimed) 2 minutes
before (baseline) and 60 minutes after occlusion. A small craniectomy
was made with a high-speed mini-drill to expose the left parietal
cortex; the dura was left intact. The LDF probe (PF 302, Perimed) was
stereotaxically placed on the exposed dura 3 mm
posterior and 5 mm lateral to the bregma; large blood vessels were
avoided. Regional CBF during occlusion was expressed as a percentage of
baseline.
Capillary plasma glucose was measured before the operative procedures
by means of a blood glucose sensor electrode (Companion 2, MediSense).
-Tocopherol in serum was analyzed by
high-performance liquid chromatography,
followed by spectrophotometry, according to a minor modification of the
methods of Lee et al10 and Nierenberg and
Nann.11
Forty-eight hours after induction of ischemia, the rats were
killed by an intraperitoneal injection of 150 mg
pentobarbital and transcardially perfused with normal saline followed
by a 4% phosphate-buffered formaldehyde solution. Brains were
carefully removed and stored in the formaldehyde solution for a minimum
of 7 days. After dehydration in a phosphate-buffered 25% sucrose
solution, coronal cryopreserved sections (25 µm) were cut and
stained with hematoxylin and eosin for histopathological evaluation.
"Infarct" was defined as the area of pallor caused by loss of
affinity for hematoxylin affecting all cell types except infiltrated
inflammatory cells.12 The areas of the infarct,
left hemisphere, and total brain were measured in each 20th section by
means of a digital image analysis system (TIM, DIFA) and
multiplied by the distance between sections to obtain the respective
volumes.
All data are expressed as mean±SD. Statistical evaluation was
performed using Student's t test and Levene's test for
equality of variances. Differences were considered to be statistically
significant at P<0.05.
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Results
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One rat died of an unknown cause 10 weeks after onset of the
vitamin E–deficient diet. Two control rats and two rats fed the
vitamin E–deficient diet died within 24 hours after MCA occlusion.
Because we could not determine infarct volume accurately in these rats,
they were excluded from further analysis, as was one control
rat in which the suture had perforated the intracranial ICA. The only
(control) rat from a different stock had an infarct volume of 252
mm3 and was excluded after an outlier test had
indicated it as an extreme value (P<0.005).
The serum level of -tocopherol in control animals was
30.9±6.6 µmol/L, which is in the range reported for rats fed
vitamin E–supplemented chow in studies of cerebral
ischemia7 and brain
trauma.13 The -tocopherol
concentration in serum of vitamin E–deficient rats was 1.5±1.1
µmol/L, which was significantly lower than that seen in control
animals (P<0.001), but somewhat higher than that in rats
maintained on vitamin E–deficient diets for 16 to 18 weeks
(0.4±0.3 µmol/L) or 8 to 10 weeks (0.6±0.5 µmol/L) in
previous studies.7 13 There was no difference in
the duration of the diet between the two groups, and the diet had no
effect on body weight or plasma glucose levels (Table 1
). Physiological
parameters are shown in Table 2. There were no differences between
groups for rectal temperature, blood gas data, heart rate, or mean
arterial blood pressure. Regional CBF in the first 60
minutes after induction of ischemia was not different between
the two groups (Table 3).
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Table 1. Age, Body Weight, and Serum Glucose Concentration of
Rats Raised on Control and Vitamin E–Deficient Diets
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Table 2. Physiological Variables of
Control and Vitamin E–Deficient Rats Before and in the First 60
Minutes After Middle Cerebral Artery Occlusion
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Table 3. Regional Cerebral Blood Flow Measured by
Laser-Doppler Flowmetry in the First Hour After Middle
Cerebral Artery Occlusion
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In both groups, most neurons, glia, and myelin in ischemic
tissue had lost their affinities for hematoxylin, and most neurons
showed eosinophilia. Occasional eosinophilic neurons with pyknotic
nuclei (red neurons) surrounded by normal tissue were found just
outside the pale area, and few neurons within the pale area showed no
abnormalities. The pale infarcts could be differentiated readily from
the surrounding normal tissue. Except for volume, there were no
differences at the cellular level between the infarcts in control rats
and those in vitamin E–deficient rats. The infarct volume was 2.2
times larger in vitamin E–deficient rats than in control rats (137±76
versus 61±19 mm3, respectively;
P=0.037). Infarct volume as a percentage of the left
hemisphere was 23±12% in vitamin E–deficient rats and 10±3% in
control animals (P=0.038).
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Discussion
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In the present study, the infarct volume after permanent
intraluminal MCA occlusion was 2.2 times larger in rats deficient in
the antioxidant vitamin E than in rats raised on a standard diet
containing 62.7 mg vitamin E per kilogram, whereas blood flow in the
MCA territory was equally reduced in both groups. This supports the
assumption that free radical formation and subsequent lipid
peroxidation are pivotal steps in the pathogenesis of focal
ischemic brain damage1 2 and points to an
important role of this vitamin as an inhibitor of oxidative
damage in this condition.
Vitamin E includes eight naturally occurring compounds in two classes,
designated as tocopherols and tocotrienols, with different
biological activities. For consistency, in this article the
amounts of all dietary compounds with vitamin E activity are expressed
as the equivalent of 1 mg of the synthetic form, racemic -tocopheryl
acetate (dl--tocopheryl acetate), which is equal to 1 IU
vitamin E.14 15 The lipid-soluble vitamin E
inhibits the peroxidation of polyunsaturated fatty acids in cellular
membranes or plasma lipoproteins.6 Dietary
deficiency of this vitamin therefore enhances the susceptibility of
biomembranes to oxidative damage.7 16 The vitamin
E requirement for the most frequently used strains of rats is 27 mg/kg
diet, when lipids compose less than 10% of the
diet.17 However, to improve fertility and health,
supplements of vitamin E to animal food have increased over the last
few years. Unfortunately, the composition of the animal diet and its
antioxidant content are often neglected by investigators. Levels of
vitamin E in animal food usually are not mentioned in articles on
experimental cerebral ischemia, even when
-tocopherol concentrations in the brain are
measured.18 19 In studies that focused on various
cerebral effects of differing levels of dietary vitamin E, the vitamin
E content of the control food usually varied between 20 and 100
mg/kg,7 13 20 21 22 with exception of up to 170
mg/kg,23 reflecting the variety in vitamin E
concentrations in the "standard" rodent diet.
Our results demonstrate that the amount of vitamin E in the standard
rodent diet provides substantial protection against focal cerebral
ischemic damage. This is in line with earlier observations that
lipid peroxidation after decapitation ischemia followed by
reoxygenation was less in vitamin E–supplemented rats
compared with control animals raised on a diet containing 20
mg/kg.7 Oral or intravenous
administration of this vitamin in the acute phase of neurological
injury has no therapeutic efficacy, because the vitamin is taken up
only very slowly by cerebral tissues.24
The availability of oxygen is a prerequisite for the peroxidation of
lipids.25 However, in contrast to the absence of
blood flow in some models of global cerebral ischemia, blood
flow reduction in focal ischemia is not absolute but ranges
from severe in the ischemic core to moderate in the
penumbra.26 27 28 In the present study,
cortical blood flow in the MCA territory during the second half hour of
ischemia was reduced to about 42% of the
preischemic baseline. Because free radicals are formed even
at very low oxygen tensions,29 reperfusion is not
a requirement for their formation.1 Hydroxyl
radical formation during both focal ischemia and subsequent
reperfusion was suggested by an increase of salicylate
hydroxylation.30 This is supported by the
present study: in the absence of recirculation the infarct volume
was larger in vitamin E–deficient rats, suggesting increased oxidative
degradation of biomembranes.
In this study, serum -tocopherol levels in vitamin
E– deficient rats were significantly decreased to 5% of the
control value. During vitamin E deficiency, -tocopherol
concentrations in the brain, spinal cord, and nerves decline more
slowly than in other tissues; after 16 weeks of deficiency,
concentrations in the brain are still 18% of control
values.22 The present study, in which rats
were maintained on a vitamin E–deficient diet for a comparable period,
demonstrates that such a concentration of vitamin E does not provide
sufficient antioxidative protection. Whether shorter periods of
deficiency and thus less decreased brain levels of vitamin E also
result in enhanced lipid peroxidation and increased infarct volume
remains to be answered in further studies. This is important, because
the rats used in most experiments are much younger than those used in
the present study.
In contrast to serum levels, brain -tocopherol levels
were not measured. In previous studies of permanent focal cerebral
ischemia in the rat, levels of 31
nmol/g19 and 40 nmol/g18
were found. In one of these studies,18
-tocopherol in ischemic tissue decreased to 63%
of the control value, but others found no evidence of consumption of
this antioxidant during permanent focal cerebral
ischemia.19
Because infarct volume was only measured 48 hours after onset of
ischemia, the present study does not provide evidence that
the effect of vitamin E is maintained for a longer period of time.
However, except for volume, no pathological differences in
ischemic tissue were found between the two groups. Therefore,
it is very unlikely that the difference in infarct volume found at 48
hours would have disappeared at a later point in time, indicating a
faster rate of infarct development in vitamin E–deficient rats as the
cause of the observed difference.
Recent prospective clinical studies have shown an inverse association
between dietary intake of vitamin E and the occurrence of
cardiovascular disease.31 32 The
effect of dietary vitamin E on outcome in acute ischemic stroke
is less extensively studied than its preventive properties. In a small
study, no difference in outcome after ischemic stroke was found
between patients with serum concentrations of vitamin E above or below
the mean value.33 A larger study applying a more
subtle subdivision of serum concentrations of this vitamin would be
required to test the effect of dietary intake of vitamin E on stroke
outcome more accurately. However, such a study will always be hampered
by the fact that its serum concentrations do not precisely reflect
those in the brain.22
In conclusion, our results support the postulate that free radical
formation and subsequent lipid peroxidation are important factors in
the pathogenesis of ischemic brain injury caused by permanent
MCA occlusion. The amount of vitamin E in the standard rodent diet
provides partial protection against this oxidative damage. The effects
of increasing supplements of vitamin E to animal food should be tested
in further studies. Researchers should be aware of a possible
confounding effect of the dietary amount of vitamin E on outcome in
animal studies of cerebral ischemia and should note the
concentration of this vitamin in the food used.
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Acknowledgments
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This study was supported by a grant from the Janivo Foundation.
We thank neuropathologist G.H. Jansen for his assistance in the
histopathological evaluation.
Received October 30, 1997;
revision received February 20, 1998;
accepted February 20, 1998.
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Editorial Comment
William I. Rosenblum, MD, Guest Editor
Department of Pathology,
Medical College of Virginia,
Virginia Commonwealth University,
Richmond, Virginia
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Introduction
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The authors convincingly demonstrate that in their model of
cerebral infarction, a diet enriched in vitamin E results in a
reduction in the size of the infarction. The reduction was not merely
statistically significant but significant in a practical sense, since
the infarct volume was reduced by 50%. It is unfortunate that the
studies were not carried out beyond a 48-hour period of survival.
Recent studies of cerebral ischemia indicate that significant
loss of neurons may occur after 48 hours. It is not yet totally clear
whether those "delayed" increments in infarct size are due only to
apoptosis or to other forms of selective neuronal necrosis, or
whether in addition there is a delayed increment in necrosis of all the
tissue elements. In the present study, infarction is defined in the
latter sense. In any case, it would be of interest to see to what
extent protection by vitamin E is manifest 1 or 2 weeks after the
ischemic event.
The authors speak of the vitamin E levels in the protecting diet as
"supraphysiological." They base this opinion on
the amount of vitamin E heretofore thought sufficient to provide the
needs of rodents. However, as is also the case in humans, there has
never been a set of objective criteria upon which to base the
assessment of a "normal" requirement for vitamin E. It may be that
evidence of frank deficiency is too crude a means for determining a
normal daily requirement. In another study, this one of mice, higher
amounts of vitamin E were required to protect the
endothelium of pial arterioles from damage that
resulted in loss of ability to produce
endothelium-derived relaxing factor following the
application of acetylcholine.1 In studies of humans, a
daily dose of 400 U has sometimes been found beneficial in the
prevention of cardiovascular disease while lesser
amounts have failed to show a benefit. The 400-U dose is far in excess
of current "daily requirements."
The proposed mechanism of vitamin E action is the scavenging of
oxygen-centered free radicals. It would be interesting to know whether
the authors can achieve an equal amount of protection with some other
radical scavenger. Regardless of mechanism, the effects of vitamin E
have obvious implications for humans. Since 400 U/d seems to be without
any adverse effects, it may be advisable to incorporate this supplement
into the daily diet.
Received October 30, 1997;
revision received February 20, 1998;
accepted February 20, 1998.
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Am J Physiol.. 1996;271:H637–H642.[Abstract/Free Full Text]
Top
Abstract
Introduction