(Stroke. 1998;29:1240-1247.)
© 1998 American Heart Association, Inc.
Postischemic Application of Lipid Peroxidation Inhibitor U-101033E Reduces Neuronal Damage After Global Cerebral Ischemia in Rats
Martin Soehle, cand. med.;
Axel Heimann, DVM;
Oliver Kempski, MD, PhD
From the Institute for Neurosurgical Pathophysiology, Johannes Gutenberg
University, Mainz, Germany.
Correspondence toUniv-Prof Dr med O. Kempski, Institute for Neurosurgical Pathophysiology, Johannes Gutenberg University, Langenbeckstr 1, 55101 Mainz, Germany. E-mail kempski{at}nc-patho.klinik.uni-mainz.de
 |
Abstract
|
|---|
Background and Purpose—The lipid
peroxidation inhibitor U-101033E was examined for effects
on cerebral blood flow (CBF), cortical tissue hemoglobin oxygen
saturation (HbSO2), and neuronal
damage.
Methods—Fifteen minutes of global cerebral ischemia was
induced by two-vessel occlusion and hypobaric hypotension. Wistar rats
(n=25) were randomized to receive vehicle (n=9) or 40 mg/kg U-101033E
(n=9) intraperitoneally during 2 hours of
reperfusion. A sham group (n=7) had neither ischemia nor
therapy. Histology was evaluated 7 days after ischemia.
Results—During late hyperperfusion (at 17 minutes),
vehicle-treated animals had a higher (P=0.044) cortical
tissue HbSO2 (72.0±1.4%) than did
U-101033E–treated animals (65.8±2.5%). Neuronal counts in the
superficial cortex layer found after 7 days correlated negatively with
rCBF (r=-0.76; P<0.001) or cortical
tissue HbSO2 (r=-0.56;
P=0.028) assessed during the late hyperperfusion phase.
U-101033E reduced neuronal damage in hippocampal CA1 from 64.3±9.2%
to 31.2±8.4% (P=0.020), as well as in the superficial
cortical layer from 53.5±14.6% to 12.8±11.7%
(P=0.046). While animals in the vehicle group had
reduced counts in all four examined cortex layers
(P<0.05 versus sham group), there was significant
cortical neuron loss in the U-101033E group in only one of four areas.
U-101033E had no effect on resting CBF or CO2
reactivity.
Conclusions—Postischemic application of U-101033E
protects hippocampal CA1 and cortical neurons after 15 minutes of
global cerebral ischemia. The results indicate that free
radical–induced lipid peroxidation contributes to reperfusion injury,
a process that can be inhibited by antioxidants such as U-101033E.
Key Words: cerebral blood flow • cerebral oxygenation • pyrrolopyrimidine • U-101033E • vasomotor reactivity • rats
|
Introduction
|
|---|
Free radical–induced
lipid peroxidation is thought to contribute to reperfusion injury after
cerebral ischemia. As soon as tissue is reperfused and hence
reoxygenated, several species of oxygen-derived free
radicals, such as superoxide anion radical, hydroxyl radical, and
hydrogen peroxide, are generated through various
pathways.1 2 3 Those radicals induce lipid
peroxidation, which is a chain reaction leading to alterations or
destruction of cell membranes and to tissue injury.
Antioxidant compounds have been developed to attenuate neuronal damage
after cerebral ischemia. The 21-aminosteroid (lazaroid)
tirilazad mesylate has been demonstrated to be a potent
inhibitor of lipid peroxidation and to reduce traumatic and
ischemic damage in a number of experimental
models.4 Tirilazad predominantly acts at
endothelial sites and has generally failed to affect
delayed neuronal damage in the selectively vulnerable CA1 region as a
result of its limited penetration into brain
parenchyma.5 Now a new generation of antioxidant
compounds, the pyrrolopyrimidines, has been developed, with a
significantly improved ability to penetrate the blood-brain barrier.6 U-101033E, a
pyrrolopyrimidine, has been shown to be a potent inhibitor
of iron-dependent lipid peroxidative neuronal injury in
vitro.6 A neuroprotective effect on hippocampal
CA1 neurons by repeated (preischemic and multiple
postischemic) application of U-101033E in the less severe
gerbil 5-minute forebrain ischemia model has been
described.6
The present study was designed to examine the effects of
postischemic treatment with U-101033E on CBF, cortical
tissue HbSO2, and
histological outcome after 15 minutes of global
cerebral ischemia.
|
Materials and Methods
|
|---|
The present study was conducted according to the current
animal protection legislation and was reviewed by the regional ethics
committee (AZ 177–07/931–20).
Animal Preparation and Hypobaric Hypotension
Twenty-five male Wistar rats (250 to 360 g body wt; strain
Crl:(WI) BR, Charles River) were premedicated with 0.5 mg atropine.
Anesthesia was introduced with ether and continued by
intraperitoneal injection of chloral hydrate
(initially 360 mg/kg body wt, afterward 120 mg/kg body wt per hour).
Animals were orally intubated and ventilated by a rodent respirator
(Harvard) during the entire experiment. The rectal temperature was
controlled at 37°C by means of a feedback-controlled homeothermic
blanket control unit (Harvard, Edenbridge GB). A loose thread was
looped around the left common carotid artery for temporary ligation
during ischemia. A polyethylene catheter was inserted into the
right common carotid artery for continuous monitoring of
arterial blood pressure by a pressure transducer (Gould
134615-50) and for blood gas analysis.
PaO2,
PaCO2, and pH were determined with
the use of an ABL 615 blood gas analyzer (Radiometer). Heart
frequency was calculated from the arterial pressure curve
by fast Fourier transformation. The peritoneum was catheterized for
fluid and drug administration. The head was fixed in a
stereotaxic frame (Stoelting), and the skull was exposed by
a 20-mm midline sagittal skin incision. Access to the brain surface was
gained through a 5x3-mm large cranial window, centered 4 mm
lateral and 4 mm caudal to the bregma. During the
craniotomy, the drill tip was cooled continuously with
physiological saline to avoid thermal injury to the
cortex. The dura was left intact (OP microscope, Zeiss). During the
experiment, the skull was continuously rinsed with 37°C
physiological saline, and skull temperature was
measured.
The lower body portion of the animals was placed in a sealable chamber,
connected to an electronically controlled vacuum pump for later
induction of hypobaric hypotension.7 To do so,
the barometric pressure within the chamber could be reduced to -30 cm
H2O (-2.9 kPa), thereby causing a pooling of
venous blood in the lower body portion of the rat.
Measurement of CBF and HbSO2
We measured lCBF using a laser flow blood perfusion monitor
(model BPM 403a, TSI) with a 0.8-mm needle probe. lCBF is expressed in
LD units because the calibration of LD to absolute flow units remains
controversial. The LD system has a reproducibly low biological
zero,8 and with the scanning technique described
below, data from individual animals and locations may be
compared.9
The local cortical tissue HbSO2 (in
percentage) was measured with the Erlangen micro–light guide
spectrophotometer (EMPHO II, Bodenseewerk Gerätetechnik). The
EMPHO II monitor consists of four modules: a light source, a
micro–light guide, a detector, and a
computer.10 11 Parallelized light from a xenon
high-pressure lamp is transmitted to the tissue surface by a central
fiber surrounded by a hexagon of six detecting fibers. Light is
scattered by the tissue, transmitted by these detecting fibers to a
fast rotating interference band-pass filter disk (502 to 628 nm), and
analyzed by a photomultiplier. The raw spectrum thus obtained
is corrected on-line with the dark spectrum and with the spectrum
obtained from excitation light reflected from a mirror at a set
distance. The response spectrum is digitized in 2-nm increments from
502 to 628 nm, and the tissue HbSO2
is calculated by an iterative best-fit procedure based on the theory of
Kubelka and Munk.12
lCBF and HbSO2 were sequentially
measured at 32 (8x4) cortical locations 300 µm apart with a
computer-controlled micromanipulator scanning technique. A fast
analog-to-digital conversion board with on-board signal processing
capacity (DAP, Microstar Laboratories) allowed us to sample data with a
running average for 8 seconds (ie, approximately 10 breathing cycles)
for each point of measurement. Therefore, one scan took 4
minutes.
Experimental Protocol
After a 20-minute control phase, 15 minutes of global cerebral
ischemia was performed by bilateral occlusion of the common
carotid arteries and MABP reduction to 42 mm Hg by hypobaric
hypotension. Reperfusion was monitored for 80 minutes. Scans were
performed for CBF at baseline conditions (minutes 3 and 14),
ischemia (minute 5) and reperfusion (minutes 1, 7, 12, 22, 30,
45, 60, and 75) as well as for HbSO2
at baseline conditions (minute 8), ischemia (minute 10), and
reperfusion (minutes 17, 35, and 65).
The animals were divided into three groups. Vehicle group animals (n=9)
received 10 mL/kg body wt of a vehicle solution (0.02 mol/L citric
acid). U-101033E group animals (n=9) received 40 mg/kg body wt of
U-101033E (Pharmacia & Upjohn Inc) in vehicle solution. Sham group
animals (n=7) had neither ischemia nor therapy and were used to
obtain data about neuronal density in the normal brain.
Therapy was administered intraperitoneally during
reperfusion according to a blinded protocol: a bolus of 10 mg/kg body
wt at minute 2, 10 mg/kg body wt per hour over 2 hours by perfusion
pump, and a final bolus of 10 mg/kg body wt after 2 hours of
reperfusion. Vehicle animals received identical volumes of vehicle
solution.
After 7 days of survival, rats were submitted to perfusion fixation
with 4% paraformaldehyde under deep
anesthesia. Histological slices were
prepared (3-µm coronal sections) and stained with cresyl violet.
Images of respective structures were obtained with the use of a light
microscope equipped with a x10 lens (Zeiss). The images were
projected onto the screen of an Amiga 2000 computer (Commodore)
with a color CCD camera (SSC-C370P, Sony) and a Genlock interface (a
special interface card). Standardized frames were superimposed over the
video image with the use of software developed in this
laboratory.13 Dimensions of the frames were
calibrated with a microscope ruler (Leitz).
Frames were adjusted over the hippocampus and cortex in a defined
manner, as schematically illustrated in Figure 1
. The frames CA1 to CA4 relate to
hippocampal areas, whereas the adjoining frames "cortex 1 to 4"
were named without relation to cortex layers. The density of cortical
neurons is regionally variable, and therefore the four equally
sized cortex 1 to 4 frames were located above the CA1 frame (Figure 1)
to achieve reproducible data. The numbers of intact neurons inside the
frames were counted for the left and right hemispheres. The mean value
of both hemispheres was calculated, and a neuronal damage index
(100%=no vital cells, 0%=mean of sham group) was determined.
View larger version (116K):
[in this window]
[in a new window]
|
Figure 1. Location of the eight frames in a coronal section
stained with cresyl violet. The frames CA1 to CA4 relate to hippocampal
sectors. The four equally sized cortex 1 to 4 frames were located above
the CA1 frame to achieve reproducible data. The number of intact
neurons was determined inside the frames for each left and right
hemisphere.
|
|
Drug Effect on Resting CBF and Vasomotor Reactivity
Additional experiments were performed to examine the effects of
U-101033E on resting CBF and vasomotor reactivity in the absence of
ischemia. To do so, three animals (300 to 320 g body wt)
were ventilated and underwent the same surgical preparations as
described above. Inspiratory and expiratory O2
and CO2 fractions were determined by a gas
analyzer (Heyer Artema MM206C). Regional cerebrovascular
CO2 responsiveness was tested by measuring rCBF
at normal and increased PaCO2 values.
The increase of CBF under the influence of elevated
PaCO2 can be best characterized by a
sigmoidal curve when viewed over a large range of
PaCO2
changes.14 15 Most authors, however, successfully
fit a straight regression line to their experimental data when the
PaCO2-CBF relationship is studied in
the range of 25 to 65 mm Hg
PaCO2. Therefore, rCBF was measured
within this range at normocapnic and hypercapnic conditions, and
cerebrovascular reactivity to CO2 was calculated
as the following ratio: %
CBF/mm Hg
PaCO2.16 17 18
After (normocapnic) baseline rCBF had been determined, vasomotor
reactivity was studied by adding 5% CO2 to the
inspired room air.18 19 20 After a steady state was
achieved, rCBF was determined. Afterward, CO2
administration was discontinued, baseline conditions were
reestablished, and a control measurement was performed 5 minutes later.
Then U-101033E was applied intraperitoneally (10
mg/kg body wt at minute 2, followed by continuous infusion of 10 mg/kg
body wt per hour) according to the treatment protocol used in the
ischemia study. rCBF measurement was performed after 15
minutes. Beginning at minute 27 during U-101033E application, vasomotor
reactivity to 5% CO2 was again determined.
Statistical Analysis
Data are expressed as mean±SEM for
physiological variables and as mean±SEM of the
median lCBF and HbSO2 from the 32
data sets from each animal. Groups were tested for normal distribution
and compared with ANOVA (Sigmastat, Jandel Scientific). Differences
were assumed statistically significant at P<0.05.
|
Results
|
|---|
The physiological variables are summarized
in Table 1. There were no statistical
differences in physiological variables and
skull temperature between the groups throughout the experiment.
View this table:
[in this window]
[in a new window]
|
Table 1. MABP, Heart Frequency, Skull Temperature, and
Arterial Blood Gas Analyses (PaCO2,
PaO2, pH) Sampled During Baseline Conditions
and Reperfusion
|
|
rCBF and HbSO2
During baseline conditions and ischemia there were no
statistical differences in rCBF or
HbSO2 between the groups.
Ischemia led to a decrease of rCBF from 50.6±7.9 to 3.4±0.7
LD units (vehicle group) and 41.3±4.0 to 4.3±0.8 LD units (U-101033E
group); both ischemic values are close to the biological
zero.
After ischemia both groups showed immediate reperfusion with
postischemic hyperperfusion, which lasted for 30 minutes
(Figure 2). The U-101033E group, however,
reached a maximum rCBF of 66.4±5.3 LD units at 7 minutes of
reperfusion and therefore earlier than the vehicle group, which showed
its maximum rCBF of 67.6±4.4 LD units only at 22 minutes. Statistical
differences in rCBF values were not found between the groups during
reperfusion.
View larger version (51K):
[in this window]
[in a new window]
|
Figure 2. Regional CBF and cortical tissue
HbSO2 of the vehicle group (n=9, therapy with
citric acid vehicle solution) and the U-101033E group (n=9, therapy
with 40 mg/kg body wt in vehicle solution). Values are mean±SEM. The
HbSO2 differs significantly
(P=0.023) at 17 minutes of reperfusion between the
groups.
|
|
The vehicle group had a cortical tissue
HbSO2 of 64.2±2.1% compared with
that of 61.3±1.3% in the U-101033E group during baseline conditions.
Ischemia reduced HbSO2 to
24.7±3.0% in the vehicle group and 28.3±2.5% in the U-101033E
group. Cortical tissue HbSO2 during
reperfusion was equal to baseline conditions except at 17 minutes of
reperfusion, when it was significantly (P=0.023) elevated in
vehicle animals. At that time, the vehicle group had a
HbSO2 of 72.0±1.4%, which was
significantly higher (P=0.044) than that in the U-101033E
group (65.8±2.5%) (Figure 2
).
Histological Outcome
Two vehicle-treated animals and one U-101033E–treated animal died
on postischemic day 2. Treatment with the pyrrolopyrimidine
significantly (P=0.046) reduced neuronal damage in the
superficial cortical layer (cortex 1) from 53.5±14.6% in the vehicle
group to 12.8±11.7% in the U-101033E group (Figure 3). Animals in the vehicle group had
significant neuronal damage in all cortex layers compared with the sham
group. The U-101033E group, however, had no reduced neuronal counts in
the cortex, except for the cortex 3 window.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 3. Neuronal damage in the cortex of the vehicle (n=7)
and the U-101033E group (n=8, 40 mg/kg body wt U-101033E) 7 days after
15 minutes of global cerebral ischemia. Cortex 1 through cortex
4 are four equally sized areas (0.07 mm2 each), with
cortex 1 most superficial and cortex 4 adjacent to white matter.
Neuronal damage of 0% corresponds to the mean neuronal density of the
sham group (n=7) that had no ischemia. 100% damage would
indicate loss of all neurons. Values are mean±SEM.
*P<0.05 vs sham group.
|
|
The most pronounced damage of all examined areas was found in the CA1
region, which is known to be selectively vulnerable to
ischemia. U-101033E significantly (P=0.020) reduced
the neuronal damage in this area from 64.3±9.2% in vehicle animals to
31.2±8.4% in treated animals (Figure 4). Other hippocampal areas had less
damage and therefore less reduction of neuronal death by U-101033E,
which was not significant.
View larger version (27K):
[in this window]
[in a new window]
|
Figure 4. Neuronal damage in the hippocampus of the vehicle
(n=7) and the U-101033E group (n=8, 40 mg/kg body wt) 7 days after 15
minutes of global cerebral ischemia. Neuronal damage of 0%
corresponds to the mean neuronal density of the sham group (n=7) that
had no ischemia. 100% damage would indicate loss of all
neurons. Values are mean±SEM.
|
|
Correlations Between Neuronal Counts and rCBF or
HbSO2
Neuronal counts in the superficial cortical layer significantly
correlated with rCBF (r=-0.76; P<0.001) (Figure 5a) and tissue
HbSO2 (r=-0.56;
P=0.028) (Figure 5b) measured after 22 or 17 minutes,
respectively, of reperfusion. This means that a high rCBF or a high
tissue HbSO2, respectively, during
the late hyperperfusion phase was followed by a low number of surviving
neurons. At those time points, animals in the vehicle group had both an
elevated rCBF and a significantly increased
HbSO2 compared with the
U-101033E–treated animals.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 5. Correlation between neuronal counts in the
superficial cortical layer and rCBF (a) or
HbSO2 (b) assessed during late
hyperperfusion.
|
|
Drug Effect on Resting CBF and Vasomotor Reactivity
Application of U-101033E affected neither resting rCBF nor
vasomotor reactivity to CO2, as examined in the
second study. Inspiration of 5% CO2
significantly increased rCBF from 38.6±12.2 to 64.7±13.7 LD units
(P=0.014) before therapy and from 37.4±9.0 to 70.1±13.5 LD
units (P=0.041) during therapy, as shown in Table 2. CO2 reactivity
was 4.6±1.5% CBF/mm Hg PaCO2
before and 4.5±1.1% CBF/mm Hg
PaCO2 during therapy
(P=NS).
|
Discussion
|
|---|
Hypobaric Hypotension
Hypobaric hypotension has been shown to be an excellent method to
reduce arterial blood pressure.7 Any
desired MABP level below the physiological MABP is
adjustable by tuning the electronically controlled vacuum
pump.8 MABP was reduced to 42 mm Hg, which
is below the lower autoregulation threshold that has been found to be
approximately 50 mm Hg in Wistar rats.8
Combination of hypobaric hypotension and two-vessel occlusion led to a
decrease of rCBF to 4 LD units, which is close to the biological zero.
Therefore, temporary global ischemia may be induced without
heparinization, bleeding, or additional occlusion of the vertebral
arteries.
Laser Doppler Scanning
LD flowmetry allows reliable, noninvasive, and
continuous recordings of CBF with a high temporal
resolution.21 22 CBF assessment by conventional
single-spot LD is highly dependent on the localization of the LD probe
because of its small spatial resolution (1
mm3). The scanning technique used in the
present study helps to overcome this disadvantage and allows
comparison of LD data from individual animals. The accuracy of repeated
scans with the use of a stepping motor-driven micromanipulator has been
found to be excellent.8 9 23 The number of
measurements necessary to obtain rCBF by local LD recordings
has been evaluated, revealing that sample sizes above n=25 are
necessary to obtain reliable information on
rCBF,9 a number well surpassed in the present
experimental paradigm. Here, the scanning technique was expanded by
including HbSO2 assessment from
identical locations, a procedure made possible by the similar sampling
volumes of both measurement techniques.
Erlangen Micro–Light Guide Spectrophotometer
The Erlangen micro–light guide spectrophotometer (EMPHO II)
allows noninvasive and continuous measurement of cortical tissue
HbSO2. The technique has been used in
various organs,10 including the
brain.11 24 25 The
HbSO2 values collected by this
spectrophotometer under control conditions are very well comparable to
data from other authors: Nakase et al11 found a
cortical tissue HbSO2 between 50%
and 60% with the same technique, and Watanabe et
al26 measured
HbSO2 values of 50% to 70% over
capillary regions using a microreflectometric system.
Histological Outcome
The results show that 40 mg/kg body wt of the
pyrrolopyrimidine U-101033E can protect hippocampal CA1 and cortical
neurons from reperfusion injury after 15 minutes of global cerebral
ischemia. This is consistent with the presumed role of
oxygen radical–induced lipid peroxidation in postischemic
neuronal damage: As soon as reperfusion after cerebral ischemia
begins, oxygen-derived free radicals, such as superoxide anion
radical27 28 29 and hydroxyl
radical,30 are generated. This is accompanied by
an increase in lipid peroxidation,31 32 33 34 which
correlates with the amount of free radical
generation.35 For the 21-aminosteroid
(lazaroid) tirilazad, a correlation has been demonstrated between
attenuation of oxygen radical levels and/or lipid peroxidation and the
neuroprotective effect.4 The pyrrolopyrimidine
U-101033E, examined in the present study, has been shown to be a
potent inhibitor of iron-dependent lipid peroxidation in
vitro,6 and the aromatized analogue U-104067F has
been shown to reduce lipid peroxidation and to attenuate
postischemic neuronal injury in the 5-minute gerbil
forebrain ischemia model (J.A. Oostveen et al, unpublished
data).
In a dose-response study using the 5-minute gerbil forebrain
ischemia model, Hall et al6 reported that
5x10 mg/kg body wt U-101033E ameliorated CA1 neuronal damage if
applied preischemically and 2, 24, 48, and 72 hours
after ischemia. In addition, they concluded that the
therapeutic window is at least 4 hours after ischemia since they had
found that a dosage of 5x30 mg/kg body wt U-101033E was effective with
a delayed initial dosing at 4 hours after ischemia. In our
study neuronal damage in hippocampal CA1 and cortex was significantly
reduced by application of only 40 mg/kg body wt during 2 hours of
reperfusion without any preischemic treatment.
Pyrrolopyrimidines are effective in focal ischemia as
well: U-101033E reduces infarct size in mice after permanent middle
cerebral artery occlusion5 and in rats after
temporary middle cerebral artery occlusion.36
Correlations
In this study a high rCBF or a high cortical tissue
HbSO2 in the late hyperperfusion
phase correlated with a low number of surviving cortical neurons 7 days
after global cerebral ischemia. This is consistent with
a positive correlation between reoxygenation level and
severity of neuronal damage in a rat model of 20-minute global cerebral
ischemia.37 Mickel et
al38 reported that exposure of gerbils to 100%
oxygen atmosphere after 15 minutes of global cerebral ischemia
resulted in increased lipid peroxidation and increased mortality.
There is an extensive inverse relationship between oxygen
availability and consumption, particularly during
postischemic hyperperfusion: oxygen availability increases
because of hyperperfusion, and oxygen consumption decreases because of
the initially reduced postischemic
metabolism.39 Therefore, enough
oxygen is available, which is presumably accompanied by an enhanced
generation of oxygen-derived free radicals and therefore increased
neuronal death.
CBF and HbSO2
All three animals that died showed a pathological
reperfusion pattern. Two animals (one vehicle-treated animal and one
U-101033E–treated animal) showed the no-reflow
phenomenon,40 which could explain the increased
mortality. The third, a vehicle-treated animal, had a continuously
increasing rCBF during reperfusion, whereas the MABP remained constant.
This animal reached a hyperperfusion that did not cease even at the end
of the 80-minute reperfusion phase, presumably because of a loss of
cerebrovascular tonus regulation.
Superoxide is generated after 20 minutes of global cerebral
ischemia, with a maximum between 10 and 15 minutes of
reperfusion,29 and produces reversible dilatation
of cerebral arterioles.41 This could explain why
vehicle-treated animals had a higher rCBF than U-101033E–treated
animals at 22 minutes of reperfusion, which was accompanied by a
significantly elevated HbSO2 at 17
minutes of reperfusion. This is in accord with reduced survival of
cortical neurons, as shown by the negative correlation between
HbSO2 and neuronal counts in the
cortex.
Drug Effect on Resting CBF and Vasomotor Reactivity
The CO2 responsiveness we found by
using the LD technique (4.6% CBF/mm Hg
PaCO2) is comparable to that in data
determined in rats with other techniques (5.5% with helium
clearance42 or 3.9% with mass
spectroscopy17). We found neither resting rCBF
nor CO2 responsiveness to be affected by
U-101033E. Therefore, we conclude that the differences in
postischemic rCBF and
HbSO2 between vehicle and treated
animals are more likely due to indirect mechanisms, such as scavenging
of vasodilating oxygen-derived free radicals, than to a direct vascular
effect of the pyrrolopyrimidine.
Conclusions
In conclusion, this is the first study to examine the
effects of U-101033E on cortical blood flow and cortical tissue
hemoglobin saturation in a global cerebral ischemia model,
demonstrating that the lipid peroxidation inhibitor
U-101033E can protect hippocampal CA1 and cortical neurons from
reperfusion injury if applied postischemically.
Preischemic or additional treatment on the days after
ischemia is not necessary to achieve a neuroprotective effect.
Our results are in agreement with the concept that free
radical–induced lipid peroxidation contributes to reperfusion injury,
a process that can be inhibited by antioxidant compounds such as
U-101033E. During the late hyperperfusion period, a high rCBF and a
high HbSO2 correlate with a low
number of surviving cortical neurons. This is consistent with
results that high postischemic oxygenation
enhances lipid peroxidation38 and leads to
increased neuronal damage.37 Since U-101033E
reduces postischemic neuronal damage in
focal6 36 as well as in global
ischemia,6 it seems to be a promising
candidate for further studies of stroke therapy.
|
Selected Abbreviations and Acronyms
|
|---|
| CBF, lCBF, rCBF |
= |
cerebral blood flow, local CBF, regional CBF |
| HbSO2 |
= |
hemoglobin oxygen saturation |
| LD |
= |
laser Doppler |
| MABP |
= |
mean arterial blood flow |
|
|
Acknowledgments
|
|---|
This study was supported in part by a grant from Johannes
Gutenberg University, Mainz, Germany (M.S.). The authors would
sincerely like to thank Dr Edward Hall from Pharmacia & Upjohn for the
supply of the pyrrolopyrimidine U-101033E; Fatemeh Kafai and Monika
Westenhuber for secretarial assistance; and Andrea Schollmayer, Michael
Malzahn, and Laszlo Kopacz for excellent technical help and support.
The data presented are part of the doctoral thesis of one of
the authors (M.S.).
Received November 19, 1997;
revision received March 3, 1998;
accepted March 20, 1998.
|
References
|
|---|
1.
Ikeda Y, Long DM. The molecular basis of brain
injury and brain edema: the role of oxygen free radicals.
Neurosurgery. 1990;27:1–11.[Medline]
[Order article via Infotrieve]
2.
Traystman RJ, Kirsch JR, Koehler RC. Oxygen radical
mechanisms of brain injury following ischemia and reperfusion.
J Appl Physiol. 1991;41:1185–1195.
3.
Phillis JW. A 'radical' view of cerebral
ischemic injury. Prog Neurobiol. 1994;42:441–448.[Medline]
[Order article via Infotrieve]
4.
Hall ED, McCall JM, Means ED. Therapeutic potential of
the lazaroids (21-aminosteroids) in central nervous system trauma,
ischemia and subarachnoid hemorrhage. Adv
Pharmacol. 1994;28:221–268.
5.
Hall ED, Andrus PK, Smith SL, Oostveen JA, Scherch HM,
Lutzke BS, Raub TJ, Sawada GA, Palmer JR, Banitt LS, Tustin JS, Belonga
KL, Ayer DE, Bundy GL. Neuroprotective efficacy of
microvascularly-localized versus brain-penetrating antioxidants.
Acta Neurochir (Wien). 1996;66(suppl):107–113.
6.
Hall ED, Andrus PK, Smith SL, Fleck TJ, Scherch HM,
Lutzke BS, Sawada GA, Althaus JS, von Voigtländer PF, Padbury GE,
Larson PG, Palmer JR, Bundy GL. Pyrrolopyrimidines: novel
brain-penetrating antioxidants with neuroprotective activity in brain
injury and ischemia models. J Pharmacol Exp
Ther. 1997;281:895–904.[Abstract/Free Full Text]
7.
Dirnagl U, Thoren P, Villringer A, Sixt G, Them A,
Einhäupl KM. Global forebrain ischemia in the rat:
controlled reduction of cerebral blood flow by hypobaric hypotension
and two-vessel occlusion. Neurol Res. 1993;15:128–130.[Medline]
[Order article via Infotrieve]
8.
Heimann A, Kroppenstedt S, Ulrich P, Kempski OS.
Cerebral blood flow autoregulation during hypobaric hypotension
assessed by laser Doppler scanning. J Cereb Blood Flow
Metab. 1994;14:1100–1105.[Medline]
[Order article via Infotrieve]
9.
Kempski O, Heimann A, Strecker U. On the number of
measurements necessary to assess regional cerebral blood flow by local
laser Doppler recordings: a simulation study with data from
45 rabbits. Int J Microcirc. 1995;15:37–42.[Medline]
[Order article via Infotrieve]
10.
Frank KH, Kessler M, Appelbaum K, Dümmler W. The
Erlangen micro-lightguide spectrophotometer EMPHO I. Phys Med
Biol. 1989;34:1883–1900.[Medline]
[Order article via Infotrieve]
11.
Nakase H, Heimann A, Kempski O. Alterations of regional
cerebral blood flow and oxygen saturation in a rat sinus-vein
thrombosis model. Stroke. 1996;27:720–728.[Abstract/Free Full Text]
12.
Kubelka P, Munk F. Ein Beitrag zur Optik der
Farbanstriche. Technische Physik. 1931;11a:76–79.
13.
Stummer W, Weber K, Tranmer B, Baethmann A, Kempski O.
Reduced mortality and brain damage after locomotor activity in gerbil
forebrain ischemia. Stroke. 1994;25:1862–1869.[Abstract]
14.
Reivich M. Arterial
PCO2 and cerebral hemodynamics.
Am J Physiol. 1964;206:25–35.
15.
Olesen J, Paulson OB, Lassen NA. Regional cerebral
blood flow in man determined by the initial slope of the clearance of
intra-arterially injected 133Xe.
Stroke. 1971;2:519–540.[Abstract/Free Full Text]
16.
Bonvento G, Charbonné R, Corrèze JL,
Borredon J, Seylaz J, Lacombe P. Is 
-chloralose plus halothane
induction a suitable anesthetic regimen for cerebrovascular research ?
Brain Res. 1994;665:213–221.[Medline]
[Order article via Infotrieve]
17.
Roussel S, Pinard E, Seylaz J. The acute effects of
MK-801 on cerebral blood flow and tissue partial pressures of oxygen
and carbon dioxide in conscious and alpha-chloralose anaesthetized
rats. Neuroscience. 1992;47:959–965.[Medline]
[Order article via Infotrieve]
18.
Lee GL, Smith JJ, Hudetz AG, Hillard CJ, Bosnjak ZJ,
Kampine JP. Laser-doppler measurement of the effects of halothane and
isoflurane on cerebrovascular CO2 response in the
rat. Anesth Analg. 1995;80:696–702.[Abstract]
19.
McPherson RW, Eimerl D, Traystman RJ. Interaction
of hypoxia and hypercapnia on cerebral
hemodynamics and brain electrical activity in dogs.
Am J Physiol. 1987;253:H890–H897.[Abstract/Free Full Text]
20.
Sandor P, Komjati K, Reivich M, Nyary I. Major role of
nitric oxide in the mediation of regional CO2
responsiveness of the cerebral and spinal cord vessels of the cat.
J Cereb Blood Flow Metab. 1994;14:49–58.[Medline]
[Order article via Infotrieve]
21.
Öberg PA, Tenland T, Nilsson GE.
Laser-Doppler flowmetry: a noninvasive and continuous
method for blood flow evaluation in microvascular studies. Acta
Med Scand 1984;687(suppl):17–24.
22.
Dirnagl U, Kaplan B, Jacewicz M, Pulsinelli W.
Continuous measurement of cerebral cortical blood flow by
laser-Doppler flowmetry in a rat stroke model. J
Cereb Blood Flow Metab. 1989;9:589–596.[Medline]
[Order article via Infotrieve]
23.
Ungersböck K, Heimann A, Kempski O. Cerebral
blood flow alterations in a rat model of cerebral sinus thrombosis.
Stroke. 1993;24:563–570.[Abstract/Free Full Text]
24.
Mayevsky A, Frank KH, Nioka S, Kessler M, Chance B.
Oxygen supply and brain function in vivo: a multiparametric
monitoring approach in the Mongolian gerbil. Adv Exp Med
Biol. 1990;277:303–313.[Medline]
[Order article via Infotrieve]
25.
Höper J, Gaab MR. Effect of arterial
PCO2 on local HbSO2 and
relative Hb concentration in the human brain: a study with the Erlangen
micro-lightguide spectrophotometer EMPHO I. Physiol Meas. 1994;15:107–113.[Medline]
[Order article via Infotrieve]
26.
Watanabe M, Harada N, Kosaka H, Shiga T. Intravital
microreflectometry of individual pial vessels and capillary region of
rat. J Cereb Blood Flow Metab. 1994;14:75–84.[Medline]
[Order article via Infotrieve]
27.
Armstead WM, Mirro R, Leffler CW, Busija DW.
Postischemic generation of superoxide anion by newborn pig
brain. Am J Physiol. 1988;255:H401–H403.[Abstract/Free Full Text]
28.
Nelson CW, Wei EP, Povlishock JT, Kontos HA, Moskowitz
MA. Oxygen radicals in cerebral ischemia. Am J
Physiol. 1992;263:H1356–H1362.[Abstract/Free Full Text]
29.
Dirnagl U, Lindauer U, Them A, Schreiber S, Pfister HW,
Koedel U, Reszka R, Freyer D, Villringer A. Global cerebral
ischemia in the rat: online monitoring of oxygen free radical
production using chemiluminescence in vivo. J Cereb
Blood Flow Metab. 1995;15:929–940.[Medline]
[Order article via Infotrieve]
30.
Phillis JW, Sen S. Oxypurinol attenuates hydroxyl
radical production during ischemia/reperfusion injury
of the rat cerebral cortex: an ESR study. Brain Res. 1993;628:309–312.[Medline]
[Order article via Infotrieve]
31.
Yoshida S, Inoh S, Asano T, Sano K, Kubota M, Shimazaki
H, Ueta N. Effect of transient ischemia on fatty acids and
phospholipids in the gerbil brain: lipid peroxidation as a possible
cause of postischemic injury. J Neurosurg. 1980;53:323–331.[Medline]
[Order article via Infotrieve]
32.
Yamamoto M, Shima T, Uozumi T, Sogabe T, Yamada K,
Kawasaki T. A possible role of lipid peroxidation in cellular damages
caused by cerebral ischemia and the protective effect of
-tocopherol administration. Stroke. 1983;14:977–982.[Abstract/Free Full Text]
33.
Watson BD, Busto R, Goldberg WJ, Santiso M, Yoshida S,
Ginsberg MD. Lipid peroxidation in vivo induced by reversible global
ischemia in rat brain. J Neurochem. 1984;42:268–274.[Medline]
[Order article via Infotrieve]
34.
Hall ED, Andrus PK, Althaus JS, VonVoigtlander PF.
Hydroxyl radical production and lipid peroxidation parallels
selective post-ischemic vulnerability in gerbil brain.
J Neurosci Res. 1993;34:107–112.[Medline]
[Order article via Infotrieve]
35.
Sakamoto A, Ohnishi ST, Ohnishi T, Ogawa R.
Relationship between free radical production and lipid
peroxidation during ischemia-reperfusion injury in the rat
brain. Brain Res. 1991;554:186–192.[Medline]
[Order article via Infotrieve]
36.
Schmid-Elsaesser R, Zausinger S, Hungerhuber E,
Plesnila N, Baethmann A, Reulen HJ. Superior neuroprotective efficacy
of a novel antioxidant (U-101033E) with improved blood-brain barrier
permeability in focal cerebral ischemia. Stroke. 1997;28:2018–2024.[Abstract/Free Full Text]
37.
Halsey JH Jr, Conger KA, Garcia JH, Sarvary E. The
contribution of reoxygenation to ischemic brain
damage. J Cereb Blood Flow Metab. 1991;11:994–1000.[Medline]
[Order article via Infotrieve]
38.
Mickel HS, Vaishnav YN, Kempski O, von Lubitz D, Weiss
JF, Feuerstein G. Breathing 100% oxygen after global brain
ischemia in Mongolian gerbils results in increased lipid
peroxidation and increased mortality. Stroke. 1987;18:426–430.[Abstract/Free Full Text]
39.
Hossmann KA, Sakaki S, Kimoto K. Cerebral uptake of
glucose and oxygen in the cat brain after prolonged ischemia.
Stroke. 1976;7:301–305.[Abstract/Free Full Text]
40.
Ames A III, Wright RL, Kowada M, Thurston JM, Majno G.
Cerebral ischemia, II: the no-reflow phenomenon. Am
J Pathol. 1968;152:437–453.[Abstract]
41.
Wei EP, Christman CW, Kontos HA, Povlishock JT. Effects
of oxygen radicals on cerebral arterioles. Am J
Physiol. 1985;248:H157–H162.
42.
Dauphin F, Lacombe P, Sercombe R, Hamel E, Seylaz
J. Hypercapnia and stimulation of the substantia innominata
increase rat frontal cortical blood flow by different cholinergic
mechanisms. Brain Res. 1991;553:75–83.[Medline]
[Order article via Infotrieve]
Editorial Comment
Costantino Iadecola, MD, Guest Editor
University
of Minnesota,
Laboratory of Cerebrovascular Biology and Stroke,
Department of Neurology,
Minneapolis, Minnesota
|
Introduction
|
|---|
Since it was first suggested that free radicals are involved
in the mechanisms of ischemia/reperfusion injury,1
it has become increasingly clear that reactive oxygen species (ROS)
play a critical role in ischemic brain damage.2 3
First, cerebral ischemia and reperfusion are associated with
production of ROS in the postischemic
brain.4 Second, pharmacological agents that
inactivate ROS or that inhibit their deleterious effects,
for example, lipid peroxidation, ameliorate cerebral ischemic
damage.5 Third, transgenic mice overexpressing the
superoxide scavenging enzyme superoxide dismutase (SOD) are relatively
protected from the consequences of cerebral ischemia, whereas
mice with deletion of the SOD gene are more susceptible to
ischemic brain injury.6 7 Therefore, ROS
scavengers or inhibitors of ROS-induced lipid peroxidation
would be useful in the treatment of ischemic stroke.
Unfortunately, lipid peroxidation inhibitors, such as
21-aminosteroids, did not show much promise in the treatment of
patients with ischemic stroke, a result that can be partly
attributed to the poor brain penetration of these agents.8
Considering the strong evidence linking ROS generation to tissue damage
resulting from reperfusion, agents with good brain penetration would be
of great therapeutic value in the treatment of ischemic stroke.
Their use would be particularly valuable to attempt to limit
reperfusion injury in patients undergoing
thrombolysis.
In the accompanying article, Soehle et al demonstrate that
posttreatment with U101033E, an inhibitor of lipid
peroxidation that crosses the blood-brain barrier, reduces the delayed
hippocampal and neocortical damage resulting from 15 minutes of global
cerebral ischemia in rats. The protection was associated with a
small but significant reduction in reactive hyperemia in the
early reperfusion phase. Importantly, however, in intact rats U101033E
did not influence resting CBF and its reactivity to hypercapnia,
suggesting that direct hemodynamic effects of the drug
are unlikely to contribute to the protection. While these data provide
additional evidence implicating ROS in ischemic brain injury,
they also suggest that U101033E may be a promising candidate for future
studies in human stroke. Additional studies in which the effect of
U101033E on postischemic lipid peroxidation is tested in
vivo may help to confirm the expected mechanisms of action of this
agent. In addition, studies of cerebral ischemia in species
phylogenetically closer to humans would provide additional evidence
supporting the potential usefulness of U101033E in patients with
ischemic stroke.
|
Selected Abbreviations and Acronyms
|
|---|
| CBF, lCBF, rCBF |
= |
cerebral blood flow, local CBF, regional CBF |
| HbSO2 |
= |
hemoglobin oxygen saturation |
| LD |
= |
laser Doppler |
| MABP |
= |
mean arterial blood flow |
|
Received November 19, 1997;
revision received March 3, 1998;
accepted March 20, 1998.
|
References
|
|---|
1.
Flamm ES II, Demopoulos B, Seligman ML, Ransohoff J.
Possible molecular mechanisms of barbiturate-mediated protection
in regional cerebral ischemia. Acta Neurol Scand
Suppl.. 1977;64:150–151.[Medline]
[Order article via Infotrieve]
2.
Kontos HA. Oxygen radicals in cerebral vascular
injury. Circ Res.. 1985;57:508–516.[Abstract/Free Full Text]
3.
Chan PH. Role of oxidants in ischemic
brain damage. Stroke.. 1996;27:1124–1129.[Abstract/Free Full Text]
4.
Traystman RJ, Kirsch JR, Koehler RC. Oxygen
radical mechanisms of brain injury following ischemia and
reperfusion. J Appl Physiol.. 1991;71:1185–1195.[Abstract/Free Full Text]
5.
Hall ED, Andrus PK, Smith SL, Oostveen JA, Scherch HM,
Lutzke BS, Raub TJ, Sawada GA, Palmer JR, Banitt LS, Tustin JS, Belonga
KL, Ayer DE, Bundy GL. Neuroprotective efficacy of
microvascularly-localized versus brain-penetrating
antioxidants. Acta Neurochir Suppl (Wien).. 1996;66:107–113.[Medline]
[Order article via Infotrieve]
6.
Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen
SF, Chan PH. Attenuation of focal cerebral ischemic
injury in transgenic mice overexpressing CuZn superoxide
dismutase. Proc Natl Acad Sci U S A.. 1991;88:11158–11162.[Abstract/Free Full Text]
7.
Kondo T, Reaume AG, Huang T-T, Carlson E, Murakami K,
Chen SF, Hoffman EK, Scott RW, Epstein CJ, Chan PH. Reduction of
CuZn-superoxide dismutase activity exacerbates neuronal cell injury and
edema formation after transient focal cerebral
ischemia. J Neurosci.. 1997;17:4180–4189.[Abstract/Free Full Text]
8.
Koroshetz WJ, Moskowitz MA. Emerging treatments
for stroke in humans. Trends Pharmacol Sci.. 1996;17:227–233.[Medline]
[Order article via Infotrieve]
Top
Abstract
Introduction
