(Stroke. 1995;26:1888-1892.)
© 1995 American Heart Association, Inc.
Articles |
Reduction of Infarct Size by Intra-Arterial Nimodipine Administered at Reperfusion in a Rat Model of Partially Reversible Brain Focal Ischemia
From the Departments of Neurosurgery (J.M.R., F.C.), Neurology (E.D.-T.), and Experimental Surgery (J.M.R., F.C.), the Hospital "La Paz," and Department of Morphology (C.A.), Medical School, Autónoma University of Madrid, Spain.
Correspondence to Dr José M. Roda, c/Pedro Rico 13, 28029 Madrid, Spain.
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Abstract |
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Background and Purpose When blood flow to a brain region that has undergone an ischemic attack is reestablished, additional injury is to be expected from the reperfusion. The purpose of the study was to determine the effect of the intra-arterial injection of nimodipine at reperfusion on infarct volume in rats subjected to partially reversible focal neocortical ischemia.
Methods Two groups of Long-Evans rats with transient bilateral common carotid artery occlusion and permanent middle cerebral artery occlusion were subjected to retrograde cannulation of the external carotid artery close to the carotid bifurcation to allow the administration of isotonic saline (group 1) or nimodipine solution (group 2) just before and during reperfusion. The estimate for the actual amount of infarcted cortex was calculated by the volume ratio between the spared cortex in the infarcted hemisphere and the total cortex of the contralateral hemisphere by means of a stereological method based on the Cavalieri principle.
Results The percentage of cortex that was infarcted in control rats was 63.8±3.1%, whereas nimodipine-treated rats exhibited a significantly smaller (P<.005) percentage of infarct volume (31.3±12.7%).
Conclusions Our data show that the intra-arterial injection of nimodipine just before and during reperfusion reduced neocortical infarct volume in rats subjected to partially reversible focal cerebral ischemia.
Key Words: neuronal damage • neuroprotection • nimodipine • reperfusion • rats
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Introduction |
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We should abandon the old idea that the amount of brain tissue surviving after ischemia depends on the anoxic damage directly provoked by the ischemic event. The possibility exists that restoration of blood circulation can result in further damage in the brain. With the expanded use of thrombolytic agents in clinical stroke trials, reperfusion injury is a fact that should be taken into consideration whenever we try to reestablish blood flow to a previously ischemic part of the brain. Reperfusion injury could explain the relatively surprising fact that sometimes permanent ischemia is better tolerated than severe transient ischemia.1 2 3
According to Jenkins et al,3 rapid deleterious structural changes in the ischemic tissue develop during the initial phase of reperfusion. The elucidation of the pathophysiological mechanisms of reperfusion injury is of great importance in designing therapeutic strategies aimed at alleviating or preventing the ensuing tissue damage. It has been proposed that brain damage during reperfusion is primarily due to harmful effects of the oxygen arriving with blood recirculation; this has been called the "oxygen paradox"4 and would lead to calcium overload and formation of oxygen free radicals, which in turn may act reciprocally on each other, giving rise to a sustained vicious circle.5
This pathophysiological frame leaves a number of options for therapeutic action. Finding neuroprotective drugs that could be administered at recirculation and that effectively avoided this additional damage to the tissue would be a substantial step in the treatment of human stroke. We recently described the protective effect of intra-arterial administration of nimodipine after transient global cerebral ischemia in rats6 and showed that this drug partially prevented the deterioration of brain function. However, although we speculated that the main effect of the drug could occur during reperfusion, we were not able to rule out an effect during ischemia. The present study was undertaken to test whether intra-arterial nimodipine would prevent or diminish the tissue damage caused by reperfusion. Proving this would prove that reperfusion injury is at least partially caused by calcium overload.
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Materials and Methods |
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The study was performed on 10 young female Long-Evans rats weighing 250±25 g. The animals were obtained from B&K Universal Ltd (Granjas Jordi, St Vicens del Horts, Barcelona, Spain). Housing and care of the animals complied with the guidelines issued by the European Union (86/609/CEE). All rats were subjected to partially reversible focal cerebral ischemia and were divided into two groups: 1 (n=5) or 2 (n=5), which received either intra-arterially administered isotonic saline or nimodipine solution, respectively, at reperfusion.
Surgery
The vascular surgical procedure was a variant of that described
by Chen et al7 and Liu et al.8 The animals
were fasted overnight and were intraperitoneally
anesthetized with 2.5 mL/kg of a solution of diazepam (2
mg/mL), ketamine hydrochloride (Ketolar, 25 mg/mL), and
atropine (0.1/mL mg). The animals were allowed to breathe unassisted.
Anesthesia was prolonged when necessary with one third of
the initial dose. Body temperature was maintained at 37±0.5°C with a
heating pad servo-controlled by a rectal probe. Procedures for
permanent middle cerebral artery occlusion were as follows: after the
zygoma and squama of the right temporal bone were exposed, a small
craniectomy was made over the main trunk of the middle cerebral artery
and above the rhinal fissure. The dura was reflected, and the middle
cerebral artery was ligated with 10/0 monofilament just before its
bifurcation between the frontal and parietal branches. Those animals
that lacked a clearly distinguishable bifurcation in these two branches
were discarded. A thermistor probe was placed under the temporal muscle
and over the cerebral cortex corresponding to the middle cerebral
artery region to measure brain temperature. This was maintained at
34°C to 35.5°C by means of a lamp located over the head. The
temporalis muscle and skin were sutured in separate layers. The femoral
artery was cannulated, and blood pressure was monitored continuously
throughout the procedure. Then the common carotid arteries were exposed
through a ventral midline cervical incision. Both were clamped during a
90-minute period, after which circulation was reinitiated. Restoration
of carotid blood flow was directly observed in all cases. Femoral
artery blood gases and plasma glucose were obtained before, during, and
after the bilateral carotid occlusion. During the occlusion period, the
right external carotid artery was retrogradely cannulated with the
catheter tip placed near the origin of the internal carotid artery as
previously described.6 9 Just before recirculation, 0.5 mL
of isotonic saline (group 1) or 40 µg/kg of nimodipine in 0.5 mL
isotonic saline solution (group 2) was injected through the catheter
via the internal carotid artery. Another 0.5 mL of isotonic saline or
nimodipine solution was injected through the catheter during the
10-minute period after recirculation. Upon completion of the surgical
procedure, all rats were returned to their cages and left alone with
free access to food and water.
Tissue Processing
Twenty-four hours after releasing the ligatures on the
common carotid arteries, we reanesthetized the animals and
perfused them through the heart with a brief rinse of saline, followed
by 600 mL of 10% formalin in 0.1 mol/L phosphate buffer, pH 7.3, using
a peristaltic pump at a mean rate of 20 mL/min. The brains were removed
and kept for a few days in the same fixative.
The brain stem was removed, and the forebrain was cut in 1.08-mm-thick slices with a tissue chopper. The slices were transferred to a 1% cresyl violet solution for 3 minutes and then were briefly washed, passed through an ascending series of ethanol, and stored for 1 to 2 weeks in 10% buffered formalin. After this treatment, infarct areas could be easily defined by a markedly lower staining intensity. The slices were then washed in phosphate buffer 0.1 mol/L and placed on the stage of a stereomicroscope for volume measurements.
Morphometry
For the sake of consistency, only the cortex between
the rhinal fissure laterally and the subiculum, callosum, and
infralimbic cortex medially10 was considered. The
measuring procedure was based on the stereological principle of
Cavalieri11 12 and consisted of the following steps:
1. The rostral surface of all slices was used. There were between 10 and 13 sections per animal, which was enough to considerably decrease the coefficient of error for the estimates (see below).
2. A quadratic lattice of points, each representing an area unit [a(p)] of 1.28 mm2, was placed randomly on each slice, and the number of points hitting the desired target area was counted at a magnification of x16.
3. The absolute area of a selected structure (intact or infarcted cortex of the right hemisphere, total cortex of the left hemisphere) in a section is
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where P(str) is the number of test points that hit any part of the specified structure projected on the test grid. Since this number is dimensionless, a(str) will be expressed in the same units as a(p) (in square millimeters).
4. The total volume of each structure is now estimated by the following formula:
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where 
is the mean section thickness (1.08
mm), and 
P(str) is the sum of all points hitting that
structure. If is expressed in millimeters,
then V will give cubic millimeters. No corrections for shrinkage
were made.
5. The precision of the volume estimate for each brain was determined by computing the coefficient of error for the estimates, which is a function of two independent factors: the variance of the point count for each section (the so-called nugget effect) and the variance of the areas for all sections (with the caveat that they be measured under systematic random sampling). Detailed accounts of the principles and the procedures for calculating coefficient of error have been published.13 14 15
The volumes of the left (intact) cortex and of the infarcted and spared portions of the right (ischemic) cortex were expressed in absolute terms (cubic millimeters). Also, the ratio between the spared cortex of the damaged hemisphere and the whole cortex of the contralateral hemisphere was computed, and this value was chosen to detect differences in the amount of cortex that was damaged by the infarct, according to our previous results.12
Statistical Analysis
The statistical analysis of the data for the two groups
was performed with the use of a statistical package
(BMDP, 1993). Values are presented as mean±SD.
Student's t test and Mann-Whitney U test were
used to compare the ratio between the spared cortex of the damaged
hemisphere and the whole cortex of the contralateral hemisphere in both
groups of animals. Since the results in relation to significance were
similar in both tests, only the figures of the Student's t
test will be presented. For analysis of
physiological parameters, a two-way
ANOVA with repeated measures was used. We considered a value of
P<.05 significant.
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Results |
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Physiological parameters, including mean arterial blood pressure, arterial blood gases, and plasma glucose, are summarized in Table 1
. Mean arterial pressure during
reperfusion showed a significant decrease of approximately 12% in
nimodipine-treated animals (group 2) with respect to untreated
animals (group 1). There were no significant differences in the rest of
the variables between groups. All rats showed a weight loss of
approximately 10% in the recirculation period.
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There were easily identifiable areas of infarction within the territory
of the occluded middle cerebral artery. The borders of the infarcted
territory were in general sharp and clearly discernible (Fig 1). Based on the results of our previous
study,12 the ratio between the spared cortex of the
right damaged hemisphere and the entire cortex in the left
contralateral hemisphere is the best index to detect differences in the
amount of cortex that was damaged by the infarct, regardless of the
intensity of the accompanying edema (Table 2). The
percentage of cortical infarction was significantly reduced in
nimodipine-treated rats compared with that in control rats (Fig 2; P<.005).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurements of the amount of infarcted brain tissue after focal ischemia are usually obtained from direct estimates of the infarct size. However, even though it is almost universally used, this method always overestimates the actual size of the infarct because of the accompanying edema, giving inaccurate results. In the last few years different authors have proposed using indirect estimations of the volume of the infarct that relate the volume of the cortex spared in the damaged hemisphere to the volume of the cortex in the contralateral hemisphere.16 17 18 In a recent study we proposed that the best estimate for the actual amount of infarcted tissue was given by the volume ratio between the spared cortex of the infarcted hemisphere and the total cortex of the contralateral hemisphere.12 This estimate was obtained by unbiased stereological methods, which have also proved to be highly efficient to compute brain volumes.12 19 20 We want to stress the importance of infarct quantification by indirect methods to avoid the error introduced by the accompanying edema and decrease the observed interanimal variance.
Different authors have reported on the beneficial effect of nimodipine or nicardipine (another dihydropyridine calcium blocker) on recirculation in global cerebral ischemia6 21 22 as well as in partly reversible focal cerebral ischemia.23 24 Nevertheless, other authors have failed to show such effects of nimodipine (administered before and/or after ischemia) either in global cerebral ischemia resulting in selective neuronal necrosis25 26 or in focal cerebral ischemia.27 28 The large variability of techniques, animal species or strains, morphometric methods, etc, makes it almost impossible to find convincing explanations for these remarkable discrepancies of results among authors. A wide agreement on a set of experimental and methodological conditions would certainly propel our current knowledge of the effects of therapeutic interventions in the various types of brain ischemia.
Until now, nimodipine has been administered before, during, or after ischemia by the intravenous route, but it had never been administered at the very moment of reperfusion or by an intra-arterial route. Prolonged intravenous administration of nimodipine starting before ischemia reduced infarct size by 20% to 60%,29 but postischemic administration had no effect on infarct size.28
Intra-arterial injection of different pharmacological substances during cerebral ischemia to impregnate the ischemic tissue6 9 makes it possible to act on brain structures at the very moment of reperfusion and in the following moments while recirculation is established. The clear benefit of delivering a neuroprotective agent with this method is that the drug reaches its target a few seconds before reperfusion starts, thus keeping the brain from being unprotected at any moment against the potential danger of reperfusion injury. Furthermore, intracarotid administration permits nimodipine to reach the ischemic area more quickly and at a higher concentration than could ever be practically achieved by the intravenous route.
According to the results of the present study, which show a decrease in the amount of infarct volume when nimodipine is intra-arterially administered at reperfusion, it seems that there is a clear effect of the drug acting just before and during recirculation and that this effect could be even greater than the effect during the period of ischemia. A high concentration of nimodipine might reach the peripheral penumbra circumscribing the central core of infarction. Siesjö30 defined the penumbra area as the periphery of the occluded middle cerebral artery territory that is nourished by collateral vessels emanating from the anterior and posterior cerebral arteries, as well as the perifocal tissues that can be salvaged by pharmacological agents or by prompt reperfusion. In the present study nimodipine could play the role of a neuroprotective drug through a vasodilatatory action31 32 in the penumbra region, and having reached this area through the previously mentioned collateral vessels via the circle of Willis, it would increase the local blood flow.
Moreover, nimodipine can help the ischemic cerebral tissue to withstand reperfusion injury through a direct neuroprotective effect on ischemic neurons. In vitro experiments have shown that mitochondria are more susceptible to damage caused by oxygen free radicals whenever the intracellular ionic calcium concentration is high.33 Cytosolic free calcium increases following reperfusion after severe cerebral ischemia,34 35 36 and this increment during reflow may have been reduced in the present study by nimodipine, as has been demonstrated in cats by Uematsu et al23 and Greenberg et al.24 By reducing the increased ionic calcium concentration, the intra-arterially administered nimodipine would block the free radical attack implicated in reperfusion injury.
In conclusion, nimodipine has reduced the size and extent of cerebral infarction in the present study either by acting directly on the metabolic disturbances, which may lead to delayed neuronal death, by improving the postischemic recirculation in the peripheral parts of the middle cerebral artery, or by both mechanisms together.
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Acknowledgments |
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This study was supported by grant FIS 93/1101. The authors wish to thank Rosario Madero for assistance in analysis of the data and Carol F. Warren for linguistic assistance.
Received December 6, 1994; revision received June 16, 1995; accepted June 20, 1995.
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