(Stroke. 1997;28:2545-2552.)
© 1997 American Heart Association, Inc.
Articles |
Differential Hydroxylation of Salicylate in Core and Penumbra Regions During Focal Reversible Cerebral Ischemia
From the Departments of Neurology (N.J.S., J.P.B.) and Neurological Surgery (N.J.S., A.-L.K., H.Y., N.F.K., K.S.L.) and the Virginia Neurological Institute (N.F.K.), University of Virginia, Charlottesville, Va.
Correspondence to Nina J. Solenski, MD, Department of Neurology, Health Sciences Center, Box 394, University of Virginia, Charlottesville, VA 22908. E-mail njs2j{at}virginia.edu.
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Background and Purpose Free radical-mediated damage during and/or after cerebral ischemia is thought to participate in the elaboration of stroke-related injury. To elucidate the role of this mechanism in cerebral damage, the study presented herein sought to clarify the spatial and temporal features of the free radical response to transient ischemia. With use of a reproducible model of in vivo focal ischemia/reperfusion, the time course of salicylate hydroxylation was measured in ischemic core and penumbra regions.
Methods Transient focal cerebral ischemia was produced in Sprague-Dawley rats by occluding both carotid arteries and one middle cerebral artery for 3 hours, followed by reperfusion. Cerebral reperfusion was confirmed by visual inspection and iodo[14C]antipyrine autoradiography. A microdialysis probe was placed stereotactically in either the ischemic core or ischemic penumbra of the frontoparietal cortex; the probe was perfused with salicylate, and dialysate samples were analyzed by high-performance liquid chromatography for salicylate hydroxylation products.
Results Salicylate hydroxylation was significantly increased during ischemia and was further increased during 6 hours of reperfusion in the penumbra compared with sham controls. In comparison, a delayed increase in hydroxylation was observed within the ischemic core region only after 3 hours of reperfusion.
Conclusion A differential generation of salicylate hydroxylation occurs in core and penumbra regions in association with focal ischemia/reperfusion of the rat neocortex. The early and progressive response in the penumbra suggests that free radical mechanisms may be continuously active in the aggravation of injury in the ischemic penumbra during ischemia and reperfusion. In contrast, the relatively delayed onset of hydroxylation in the core region indicates that this mechanism participates primarily in the late stages of ischemic injury in densely ischemic tissue. These findings are consistent with the concept that the role of free radicals in cerebral injury may differ qualitatively and/or quantitatively in areas of total and partial cerebral perfusion.
Key Words: cerebral ischemia, transient • cerebral ischemia, focal • oxygen radical • reperfusion • rats
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The toxic effect of oxygen free radicals on the cerebral vasculature and surrounding brain parenchyma after acute cerebral ischemia and reperfusion is diverse and well documented.1 2 3 4 5 6 Free radical species are capable of directly injuring endothelial and smooth muscle cells, which can lead to deleterious secondary effects including increased platelet aggregation, increased vascular permeability, or cellular death.7 8 9 10 11 12 In cerebral vessels, oxygen free radical-mediated impairment of the blood-brain barrier can result in potentially life-threatening cerebral edema. Indirect evidence of the neurotoxic effects of free radicals is extensive and includes increased levels of conjugated dienes and other byproducts of lipid peroxidation, elevation and depletion of endogenous free radical scavengers, alterations of DNA, and oxidation of proteins. Only recently, through technical refinements, have in- vestigators been able to directly observe reactive oxygen species in situ. Studies measuring reactive oxygen species in animal models of both global and focal cerebral ischemia/reperfusion have used sophisticated techniques such as electron paramagnetic resonance, on-line chemiluminescence, and the use of free radical trapping agents, including salicylate.13 14 15 16 17 In the latter technique, the aromatic hydroxylation of salicylate is often interpreted as a marker of hydroxyl radical (·OH) generation. Because of these techniques, an initial understanding of the magnitude, temporal course, and origin of the oxygen free radical species in situ during reversible focal cerebral ischemia is beginning to emerge. Recent efforts by Morimoto et al17 and other investigators16 have demonstrated the generation of free radicals in rat models of global and focal cerebral ischemia, using the microdialysis and salicylate technique. Additional research defining the types of radical species that are generated, where they are generated, and the potential reversibility of their deleterious effects is essential to the development of neuroprotective strategies targeting free radical-induced injury.
In an effort to clarify these questions, the goal of the present study was to define the temporal and spatial characteristics of oxygen free radical generation during focal ischemia/reperfusion of the neocortex. Levels of hydroxylated salicylate were measured using a reproducible model of focal cerebral ischemia in the rat combined with regional brain microdialysis. The hypothesis that oxygen free radical species are formed during the early reperfusion phase of reversible ischemia, coincident with the reintroduction of oxygen to the brain tissue, was examined.
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All experimental protocols were approved by the Animal Research Committee of the University of Virginia, and all animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care.
Rat Surgery—Focal Reversible Cerebral Ischemia
Reversible focal neocortical ischemia was produced in
normotensive Sprague-Dawley male rats, using a reproducible model of
three-vessel occlusion (3VO18 19 ). Briefly, this method
consists of the following: Adult Sprague-Dawley male rats (Hilltop Lab
Animal Inc.) weighing 250 to 300 g were orotracheally
intubated, anesthetized with 4% halothane in O2,
and mechanically ventilated with a 1.5% halothane/oxygen mixture
(WECO; Harvard Rodent Ventilator, Harvard Instrument Co.) The common
carotid arteries were ensnared bilaterally with reversible nylon
ligatures. On the left side of the skull, a small (2-3 mm)
craniotomy was made and a small slit in the dura was
created; the left middle cerebral artery (MCA) was identified, and a
small segment distal to the lenticulostriate branches was isolated from
the surrounding meningeal tissue. A microsurgical clip (Sundt AVM
Microclip, Codman) was carefully applied to the MCA, followed by
immediate tightening of nylon snares around the carotid arteries. After
3 hours of ischemia, cerebral circulation was reinstituted by
removing the microaneurysmal clip and loosening the common
carotid snares. Mean arterial blood pressure, heart rate,
and brain and rectal temperature were continuously monitored. Each
hour, a 300-µL aliquot of blood was drawn from the left femoral
artery catheter for the analysis of
PaO2, HCO3,
PaCO2, and pH (278 Blood Gas Analyzer,
CIBA-Corning). Sham operations were performed in an identical manner to
the surgery described above, except that the microsurgical clip was not
applied and the carotid snares were not tightened. Each experimental
group consisted of four to six rats per experiment.
Confirmation and Measurement of Infarction Area
Analysis of the area of cerebral infarction was
performed in animals sacrificed 24 hours postreperfusion. In these
studies, the rat was anesthetized, intracardially perfused with
normal saline, and decapitated, and the brain was quickly removed.
During the removal of the brain, a careful visual inspection of the
insertion point of the microdialysis probe was made to provide initial
confirmation of the stereotactic placement into either the
core or penumbral region. To remove the probe from the brain, the
entire skull and brain (with the attached probe) were placed into a
stereotaxic frame and the skull was circumferentially cut
and carefully elevated in a vertical plane away from the underlying
brain tissue to prevent artifact damage to the parenchyma. Serial
coronal brain sections (2 mm in thickness) were prepared and were
stained with 2% 2,3,5-triphenyltetrazolium
chloride (TTC)20 (Sigma Chemical Co.). TTC, a colorless
salt, is reduced to form an insoluble red formazan product in the
presence of a functioning mitochondrial electron transport chain. Thus,
the infarcted region lacks staining and appears white, whereas normal,
noninfarcted tissue appears pink. This color distinction facilitates
subsequent measurement of the volume of infarcted tissue using a
digital image analysis system (Image-1, Universal Imaging Co.)
Previous studies performed in our laboratory confirmed that the
cerebral infarction produced by this model results in a cortical
infarction that is reproducible with respect to both its size and
location.18 21 22 23 24 As mentioned previously, the occlusion
of the MCA is distal to the origin of the lenticulostriate arteries,
which typically restricts the area of infarction to the neocortex.
Measurement of Iodo[14C]antipyrine
([14C]IAP) Concentrations in Cerebral Tissue
The tissue concentration of [14C]IAP was measured
by a modification of the autoradiographic technique
described by Sakurada et al.25 A femoral vein catheter was
inserted in the right limb of an anesthetized rat, and the
three-vessel occlusion surgery was performed as detailed above. A
100-µCi/kg bolus of iodo[14C]antipyrine (Amersham) was
quickly injected via the femoral vein catheter into control animals or
into ischemic animals at either 3 hours of ischemia (no
reflow), 15 minutes of reperfusion, or 3 hours of reperfusion. At
exactly 1 minute after venous injection, the rat was decapitated and
the brain was quickly removed and frozen in chilled isopentane. The
brain was either stored at -80°C or serially sectioned (20
µm) with a cryostat (-25°C). Serial brain sections and
14C-labeled standards (on glass slides, .04-400 nCi/mg
range; Amersham) were exposed to autoradiography film
for 72 hours (BioMax, Kodak). With use of a microcomputer imaging
device (Imaging Research Inc.), a calibration curve correlating optical
density to each known concentration of 14C was calculated
using the densitometric measurement. Local tissue
[14C]IAP concentration in the neocortex was used to
identify the ischemic core and penumbra regions ipsilateral to
the occluded MCA. [14C]IAP concentration in microcurie
per gram was calculated from these densitometric measurements by
interpolation using the generated 14C calibration curve.
Sampling calculations are based on three separate sampling areas from
within either the penumbra or the core region of the frontoparietal
cortex of both the left (ischemic) and right
(nonischemic) cerebral hemispheres from three separate serial
brain sections. A total of six animals were analyzed.
Brain Microdialysis
Twenty-four hours before 3VO surgery, a microdialysis probe
(CMA) was stereotactically placed into the left
frontoparietal cortex of a halothane-anesthetized rat and
secured to the skull with stainless steel screws and acrylic dental
cement. Separate pilot studies were performed to determine the
stereotactic coordinates of both the core and penumbral
region by examining the patterns of [14C]IAP activity
with both TTC and hematoxylin and eosin–stained coronal rat brain
sections; coordinates of the identified areas were then determined
using a standard rat brain atlas (n=10).26 The probe was
implanted using a stereomicroscope to avoid rupturing meningeal or
cortical surface vessels. Buffered artificial cerebral spinal fluid
with 500 µmol salicylate (Sigma) was perfused through the probe
at a flow rate of 2 µL/min. Salicylate was administered in vivo
through a microdialysis probe, which was implanted into either the
severely ischemic core region or within the surrounding
ischemic penumbra region. Dialysate samples were then collected
in .5 mol/L hydrochloric acid at 20-minute intervals during a standard
experimental paradigm of 1 hour of baseline, 3 hours of
ischemia, and 6 hours of reperfusion time. Samples were
analyzed for 2,3- and 2,5-dihydrobenzoic acid (DHBA) levels by
high-performance liquid chromatography (see
below) or were stored at -80°C for future analysis.
HPLC Analytical Techniques
The hydroxylation products of salicylate, 2,3- and 2,5-DHBA,
were measured in 20-µL aliquots of dialysate using an established
high-performance liquid chromatography
electrochemical detector technique.27 Samples were
separated over a catecholamine C18 column (Alltech), were
electrochemically detected using a mobile phase consisting of 2
mmol/L 1-heptane-sulfonic acid and 1 mmol/L
ethylenediamine-tetraacetic acid (Sigma) with 5% methanol (Fisher
Scientific) at a pH of 2.9 in a 50 mmol/L (mM) sodium phosphate
buffer, and were electrochemically detected (Coulchem II; ESA). The
concentration of 2,3- and 2,5-DHBA was determined by comparison of
known nanomolar standard concentrations of 2,3- and 2,5-DHBA.
Nonspecific production of 2,3-DHBA and 2,5-DHBA generated by
the microdialysis system was measured and found to be negligible.
Statistical Analysis
The statistical analysis of within-group differences of
salicylate hydroxylation was performed using a multiple
analysis of variance test. Temporal and spatial differences
between groups for salicylate hydroxylation concentrations were
determined by a two-way analysis of variance test with multiple
comparisons performed by either Dunnett's or Tukey test.
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Regional [14C]IAP Concentrations in Cerebral Tissue
The ischemic penumbra was defined on the basis of two overlapping criteria in the present study. The first criterion for identifying the penumbra was based on previous studies using the 3VO model in which the regions sensitive to 3 hours of occlusion but not to 1 hour of occlusion were identified.18 The second criterion for identifying the penumbra was the degree of reduced blood flow in the same regions as identified in experiments using the tracer iodo[14C]antipyrine. Blood flow was reduced substantially in the core, whereas only partial reductions were observed in the penumbra. Fig 1A
is a schematic coronal
brain section illustrating the typical penumbra and core
ischemic region(s) after 3 hours of left hemispheric MCA
occlusion in the 3VO rat model. A pseudocolor-enhanced digital
autoradiographic image of a coronal brain section after
iodo[14C]antipyrine injection during the end of the third
hour of left hemispheric MCA ischemia illustrates these two
regions (Fig 1B). At the end of the ischemic period (3 hours)
but before reflow, a well demarcated area of decreased radioactivity,
localizing to the typical MCA blood flow territory, was demonstrated
ipsilateral to the MCA (left hemisphere) compared with the
corresponding region of the contralateral neocortex. The
ischemic core region where perfusion is negligible appears red
or yellow and is easily distinguished from the surrounding penumbra
demonstrated in blue enhancement (Fig 1B). The nonischemic
tissue (normal flow) appears black as seen in the contralateral
hemisphere and non-MCA territory. Densitometric measurements
demonstrated that the difference between the ipsilateral and
contralateral cortices achieved statistical significance
(P<.05). A time-dependent change in regional
[14C]IAP radioactivity was observed during
ischemia-reperfusion in both penumbra and core ischemic
regions (Fig 2). Radioactivity levels
were significantly decreased in the ischemic left cortex of
both the core and penumbra region compared with control
nonischemic levels. There was a 86% reduction in blood flow in
the ischemic core and a 48% reduction of flow in the penumbra
region. Within the first 15 minutes of reinstitution of cerebral blood
flow, regional [14C]IAP concentration within the sampling
area of both the penumbra and core regions of the left frontoparietal
cortex was significantly higher than intraischemic levels when
expressed as a percent of the control or nonischemic condition
(P<.05). After a total of 3 additional hours of
reperfusion, the levels of [14C]IAP within the
ischemic hemisphere were no longer significantly different from
the nonischemic control, indicating a reinstitution of
flow.
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Physiological Parameters
The physiological variables of mean
arterial blood pressure, heart rate, brain temperature,
rectal temperature, PaCO2,
PaO2, and pH were monitored hourly during all
experiments. The mean and the standard deviation of the mean of these
variables measured during the baseline, ischemic, and
reperfusion periods are presented in Table 1. Physiological
parameters, particularly rectal temperature, were carefully
maintained within normal accepted ranges for the rat species.
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Histopathology—TTC Staining
The presence of a consistent cerebral infarction in
ischemic animals was confirmed in all but one experimental rat;
this individual rat was, therefore, excluded from analysis. Fig 3A illustrates the external surface of a
rat brain with a typical TTC staining pattern after 3 hours of
ischemia and 24 hours of reperfusion. Occlusion of the MCA
distal to the lenticulostriate arteries infarcted substantial areas of
the frontal and parietal cortices, while sparing the caudate and
putamen region. The darkened area (Fig 3) is India ink introduced
through the microdialysis probe and indicates the
stereotactical placement of the probe within the penumbral
region (arrow "a" in Fig 3). Stereotactical placement
within the "core" ischemic region (asterisk in Fig 3) was
3 mm lateral to that of the penumbral area. Serial coronal
sectioning of the whole brain and staining with TTC demonstrate the
full extent of the infarction within the typical MCA territory (Fig 3B).
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Salicylate Hydroxylation—Core and Penumbra
The baseline (preischemic) level of salicylate
hydroxylation was observed to vary somewhat among experimental animals.
All data from individual animals were, therefore, normalized to their
baseline values and expressed as a percentage of mean baseline
concentration of 2,3-DHBA (moles/hour). The range of baseline absolute
concentration of 2,3-DHBA was 1.1 to 3.9 pmol/hour within the penumbra,
1.4 to 15.6 pmol/hour within the core, and 2.6 to 9 pmol/hour
(picomoles/hour) during the first 60 minutes of the sham surgery
experiments. Because measured values for 2,5-DHBA may reflect
microsomal p450 enzymatic hydroxylation of salicylate rather than in
situ salicylate hydroxylation, the levels of 2,5-DHBA are not
presented.28
Salicylate hydroxylation was increased substantially and significantly
in the penumbra compared with samples from the ischemic core or
a comparable region of sham-operated rats. As shown in Fig 4, an increase in 2,3-DHBA formation
within the penumbra was observed during ischemia, and a second
progressive increase was observed during the 6 hours of reperfusion. In
contrast to the penumbral region, there was no significant increase of
2,3-DHBA formation within the ischemic core throughout the 3
hours of ischemia and for the first 3 hours of reperfusion
(P>.05). The concentration of 2,3-DHBA formation within the
ischemic core was not statistically different from that
measured in the same cortical region of sham-operated animals during
the period corresponding to ischemia and 3 hours of
reperfusion. However, a significant, delayed elevation of salicylate
hydroxylation was observed within the core after the third hour of
reperfusion and continued during the subsequent 3 hours of reperfusion
(P<.05; Fig 4).
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Oxygen free radicals are postulated to play a critical role in the expression of brain injury associated with ischemia/reperfusion. A clear understanding of the precise time course over which these radical species are generated and the regional distribution of this response is, therefore, important for knowing how ischemic injury progresses. Technical advances for measuring radical species have facilitated investigations of the spatial and temporal features of the free radical response to transient cerebral ischemia. By using microdialysis techniques, recent studies have demonstrated intraischemic and postischemic elevations of hydroxyl radical (or salicylate hydroxylation) in partially perfused cerebral cortex and striatum.16 17 29 30 31 The results of the present study generally confirm these findings by demonstrating an intraischemic and postischemic elevation of salicylate hydroxylation in the penumbra of a transient focal ischemic event. The present findings also extend those of the previous studies in several ways. First, the free radical responses were shown to differ significantly in core versus penumbra regions. Second, the use of an extended time course of measurement revealed a second and prolonged phase of increased hydroxylation in the penumbra, which persists for at least 6 hours into the reperfusion phase. Third, although the core region does not exhibit an early increase in salicylate hydroxylation, a substantial, delayed increase is observed beginning several hours after reperfusion.
The elevation of 2,3-DHBA during the intraischemic period in the penumbra indicates that this region retains sufficient oxygen tension for the hydroxylation of salicylate; in contrast, the oxygen levels in the core are likely to be insufficient to sustain the intraischemic production of oxygen free radicals. It is also notable that the re-establishment of blood flow and reoxygenation of the tissue produce a gradual rather than an immediate spike (or "burst") of oxygen radical production in the penumbra. In the core, this response is delayed even longer, with the elevation of salicylate hydroxylation occurring only after a postischemic delay of several hours. The different time courses of salicylate hydroxylation in the core and penumbra in response to ischemia/reperfusion suggest that the roles played by oxygen free radicals in ischemic injury could differ in these regions. A similar analysis in sham-operated animals over the same time course confirms that this observation does not represent an artifact of the microdialysis recovery efficiency over the prolonged temporal course. The most plausible explanation for the different time courses is that the temporal evolution of free radical-related damage differs in areas of partial and complete ischemia.
The present findings indicate that salicylate hydroxylation within the penumbral region occurs as early as the first hour of ischemia. Recently, it has been demonstrated that there is a rapid rise in nitric oxide levels during the first 2 hours of ischemia, perhaps via calcium-dependent activation of nitric oxide synthase.32 33 34 35 Conceivably, nitric oxide-mediated salicylate hydroxylation could occur during acute ischemia and may involve the production of peroxynitrite anion.36 37
Recently, additional proposed mechanisms of oxygen free radical generation during acute ischemia within the penumbra include evidence supporting the role of mitochondria-mediated hydroxyl radical generation in vivo during ischemia.38 It has been postulated that during acute ischemia "reductive stress" occurs, mainly from the inability of damaged mitochondria to accept electrons at the terminus of the respiratory chain. The consequence of increased electrons is an electron leak at the proximal site on the electron transport chain with a subsequent increase in free radical formation.
The delayed phase of salicylate hydroxylation in the core could be attributable to any of several factors. One possibility is that this response reflects a later stage of the degenerative response of cells in tissue undergoing infarction. These effects could include the induction of enzymatic mechanisms, such as activation of inducible nitric oxide synthase. Another possibility is that the delayed rise in hydroxylation is related to the early beginnings of tissue infiltration of neutrophils, which are known to be capable of generating free radicals.39
It is possible that severe ischemia compromises reperfusion of the microcirculation, resulting in a "no-flow" phenomenon.40 Impairment of the microcirculation after MCA occlusion during reperfusion is well documented.40 41 42 43 44 In this setting, despite gross re-establishment of blood flow, reoxygenation would be severely limited. Recent clinical neuroimaging studies using diffusion-weighted or gradient echo magnetic resonance image45 or using positron emission tomography analysis after occlusion of the MCA also support the presence of a microcirculatory defect. Clarification of this issue will await future investigation.
It is important to recognize the limitation of microdialysis studies when interpreting the study presented herein. First, although aromatic hydroxylation of salicylate is a well established assay for measuring in vitro and in vivo ·OH production and is an accepted marker of in vivo oxidative stress in various disease states including cerebral ischemia,16 17 46 47 48 this technique requires careful consideration. Salicylate inhibits cyclooxygenase, attenuating the metabolism of arachidonic acid and potentially decreasing the formation of free radicals during acute ischemia. For this reason, the application of salicylate during cerebral ischemia may underestimate the total free radical activity in the brain interstitium. Second, despite taking great care to minimize tissue damage, intracerebral microdialysis is an invasive technique. The extent of tissue damage is usually considered to be small and depends on the size of the probe and the care taken during probe implantation and allowing for stabilization of baseline measurements.49 50 Finally, as mentioned previously, microdialysis recovery rates are dependent on a multitude of factors, including the location of the solute formation compared with the location of the probe (intracellularly versus extracellularly), the solute type, molecular weight, and charge. Despite these caveats, the ability to directly measure in vivo the temporal and spatial production of hydroxylated products as a marker for oxygen free radical production and the ability to biochemically analyze potential mediators and neuroprotectants outweigh the above-mentioned technical limitations.
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Focal ischemia/reperfusion elicits complex temporal and spatial alterations of oxygen free radical activity in the rat neocortex. The ischemic penumbra is subject to early and sustained free radical activity, whereas the densely ischemic core region exhibits a substantial delay before exhibiting free radical production. Notably, a profound burst of oxygen free radical production on reperfusion is not a prominent feature of the response to ischemia/reperfusion in this model system. These findings suggest that the role of oxygen free radical generation in the expression of ischemic injury may vary according to both the intensity of ischemic challenge and the time course of ischemic compromise.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by National Institutes of Health Grant HL49396 to KSL and by National Institutes of Health Grant 1K08NS01857 (Mentored Clinical Scientist Development Award) to NJS. The authors gratefully acknowledge the excellent assistance of both Sarah B. Hudson, BS, and Trisha Smith, BS.
Received March 31, 1997; revision received September 4, 1997; accepted September 8, 1997.
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