Huang ZG, Xue D, Preston E, Karbalai H & Buchan AM.Biphasic opening of the blood-brain barrier following transient focal ischemia: effects of hypothermia. Can J Neurol Sci 26: 298−304

Abstract

Tracer constants (Ki) for blood-to-brain diffusion of sucrose were measured in the rat to profile the time course of blood-brain barrier injury after temporary focal ischemia, and to determine the influence of post-ischemic hypothermia.
Spontaneously hypertensive rats were subjected to transient (2 hours) clip occlusion of the right middle cerebral artery. Reperfusion times ranged from 1.5 min to 46 hours, and i.v. 3H-sucrose was circulated for 30 min prior to each time point (1 h, 4 h, 22 h, and 46 h; n = 5-7 per time point). Ki was calculated from the ratio of parenchymal tracer uptake and the time-integrated plasma concentration. Additional groups of rats (n = 7-8) were maintained either normothermic (37.5 degrees C) or hypothermic (32.5 degrees C or 28.5 degrees C) for the first 6 hours of reperfusion, and Ki was measured at 46 hours.
Rats injected after 1.5-2 min exhibited a 10-fold increase in Ki for cortical regions supplied by the right middle cerebral artery (p < 0.01). This barrier opening had closed within 1 to 4 hours post-reperfusion. By 22 hours, the blood-brain barrier had re-opened, with further opening 22 and 46 hours (p < 0.01), resulting in edema. Whole body hypothermia (28 degrees C-29 degrees C) during the first six hours of reperfusion prevented opening, reducing Ki by over 50% (p < 0.05).
Transient middle cerebral artery occlusion evokes a marked biphasic opening of the cortical blood-brain barrier, the second phase of which causes vasogenic edema. Hypothermic treatment reduced infarct volume and the late opening of the blood-brain barrier. This opening of the blood-brain barrier may enhance delivery of low permeability neuroprotective agents.

Full text

298
ABSTRACT: Objective: Tracer constants (K
i
) for blood-to-brain diffusion of sucrose were measured in the rat to profile the time
course of blood-brain barrier injury after temporary focal ischemia, and to determine the influence of post-ischemic hypothermia.
Methods: Spontaneously hypertensive rats were subjected to transient (2 hours) clip occlusion of the right middle cerebral artery.
Reperfusion times ranged from 1.5 min to 46 hours, and i.v.
3
H-sucrose was circulated for 30 min prior to each time point (1h, 4h, 22h,
and 46h; n=5-7 per time point). K
i
was calculated from the ratio of parenchymal tracer uptake and the time-integrated plasma
concentration. Additional groups of rats (n=7-8) were maintained either normothermic (37.5
o
C) or hypothermic (32.5
o
C or 28.5
o
C) for
the first 6 hours of reperfusion, and K
i
was measured at 46 hours. Results: Rats injected after 1.5 – 2 min exhibited a 10-fold increase
in K
i
for cortical regions supplied by the right middle cerebral artery (p<0.01). This barrier opening had closed within 1 to 4 hours post-
reperfusion. By 22 hours, the blood-brain barrier had re-opened, with further opening 22 and 46 hours (p<0.01), resulting in edema.
Whole body hypothermia (28
o
C-29
o
C) during the first six hours of reperfusion prevented opening, reducing K
i
by over 50% (p<0.05).
Conclusions: Transient middle cerebral artery occlusion evokes a marked biphasic opening of the cortical blood-brain barrier, the
second phase of which causes vasogenic edema. Hypothermic treatment reduced infarct volume and the late opening of the blood-brain
barrier. This opening of the blood-brain barrier may enhance delivery of low permeability neuroprotective agents.
RÉSUMÉ: Ouverture biphasique de la barrière hémato-encéphalique suite à une ischémie focale transitoire: effets de l’hypothermie. Objectif:
Nous avons mesuré les constantes d’un traceur (K
i
) de la diffusion de sucrose du sang vers le cerveau chez le rat afin d’observer l’évolution des
dommages subis par la barrière hémato-encéphalique après une ischémie focale temporaire et pour déterminer les effets d’une hypothermie post-
ischémique. Méthodes: Des rats spontanément hypertendus ont été soumis à une occlusion de deux heures de l’artère cérébrale moyenne par un clip.
Le temps de reperfusion variait de 1.5 minute à 46 heures et une perfusion intraveineuse de
3
H-sucrose a été administrée pendant 30 minutes avant
chaque évaluation ponctuelle (1h, 4h, 22h, et 46h; n=5-7 par évaluation ponctuelle). La constante K
i
a été calculée à partir de l’indice de captation du
traceur par le parenchyme et de la concentration plasmatique en fonction du temps. Des groupes additionnels de rats (n=7-8) ont été maintenus soit à
la température normale (37.5
o
C) ou en hypothermie (32.5
o
C ou 28.5
o
C) pendant les 6 premières heures de la reperfusion et K
i
a été mesurée à 46 heures.
Results: Les rats qui ont reçu l’injection après 1.5 – 2 minutes présentaient une augmentation de K
i
de dix fois supérieure dans les régions corticales
irriguées par l’artère cérébrale moyenne (p<0.01). Cette ouverture de la barrière s’était refermée 1 à 4 heures post-reperfusion. À 22 heures, la barrière
hémato-encéphalique s’était réouverte, davantage à 22 et à 46 heures (p<0.01), ce qui a donné lieu à de l’œdème. L’hypothermie généralisée (28
o
C -
29
o
C) pendant les 6 premières heures de la reperfusion a empêché son ouverture, diminuant ainsi la constante K
i
de plus de 50% (p<0.05). Conclusions:
L’occlusion transitoire de l’artère cérébrale moyenne provoque une ouverture biphasique importante de la barrière hémato-encéphalique corticale dont
la deuxième phase cause de l’œdème. L’hypothermie a diminué la taille de l’infarctus cérébral et l’ouverture tardive de la barrière hémato-encéphalique.
Cette ouverture de la barrière hémato-encéphalique peut accroître la distribution d’agents neuroprotecteurs à basse perméabilité.
Can. J. Neurol. Sci. 1999; 26: 298-304
Brain capillary walls are distinguished by an endothelial cell
layer replete with tight junctions and a scarcity of fenestrae. This
blood-brain barrier (BBB) is a specialization which maintains
homeostasis of the neuronal micro-environment, limiting blood-
to-brain diffusion of hydrophilic molecules. Penetration is
largely restricted to lipophilic substances capable of directly
traversing endothelial membranes, and to hydrophilic substances
such as amino acids and glucose, for which specific membrane
carriers exist.
1,2
BBB drainage, following brain ischemia, leads
to the extravascular leakage of plasma proteins and other solutes,
resulting in an imbalance with osmotic forces drawing excess
water into the tissue (i.e. vasogenic edema).
3
Tissue swelling
ensues within the rigid confines of the skull, elevating
intracranial pressure with secondary ischemia due to
compression of microvasculature, and, ultimately, brain
herniation.
4
Biphasic Opening of the Blood-Brain
Barrier Following Transient Focal
Ischemia: Effects of Hypothermia
Z. Gao Huang, Dong Xue, Edward Preston, Hasneen Karbalai,
Alastair M. Buchan
From the Alberta Stroke Program, Department of Clinical Neurosciences, University
of Calgary, Alberta, (ZGH ,DX, HK, AMB); and the Institute for Biological Sciences,
National Research Council, Ottawa, Ontario, Canada (ZGH, DX, EP)
RECEIVED JANUARY 9, 1999. ACCEPTED IN FINAL FORM MAY 18, 1999
Reprint requests to: Alastair M. Buchan, Alberta Stroke Program, Office of Stroke
Research, Room 1162, Foothills Hospital, 1403 – 29th Street NW, Calgary, AB,
Canada T2N 2T9
EXPERIMENTAL
NEUROSCIENCES

A reproducible rodent model used to study focal brain
ischemia involves temporary (e.g. 0.5 to 4 hours, or permanent
occlusion of a middle cerebral artery [MCAO model] with
tandem occlusion of the ipsilateral common carotid artery).
5,6,7
Temporary MCAO enables the study of both positive and
negative aspects of post-ischemic reperfusion, which occurs
clinically, both spontaneously and therapeutically, with the
advent of r-tPA, newly licensed as a thrombolytic agent for
ischemic stroke in the first three hours. Restoration of blood
flow, if early enough, may offer the advantage of reducing
neuronal damage and limiting infarct extension and enhances
potentially cytoprotective drug delivery. These benefits may be
undermined by reperfusion injury to the microvasculature,
compromising BBB function, exacerbating edema formation and
the inflammatory developments that follow.
2,3,4
Dynamic
changes in BBB permeability, which follow temporary MCAO,
are therefore of critical importance.
The objective of this study was to delineate the time course
and intensity of the BBB opening after reversible MCAO
ischemia in the rat using a method involving a removable,
atraumatic clip.
6
Post ischemic changes in the BBB were studied
using a modification
8
of a radiotracer methodology, which
quantifies even minor degrees of BBB opening with a high
degree of sensitivity.
9,10
The radiotracer method used in this
study is based on a 2-compartment (plasma/brain) simple
diffusion model, which assumes that the amount of
3
H-sucrose,
which permeates the microvasculature into brain parenchyma,
does so in proportion to the time integral of plasma tracer
concentration. Normal BBB permeability and opening are
indexed in the ratio of parenchymal uptake relative to plasma
integral, calculated as the transfer constant, K
i
.
9,10
The effects of
delayed hypothermia
11
on the biphasic BBB opening were also
investigated.
METHODS AND MATERIALS
Male spontaneously hypertensive rats (SHR) weighing 170g
– 225g were fasted for 18 hours prior to surgery, but allowed free
access to water. Animals were initially anesthetized with 3%
halothane, and subsequently maintained on 1 – 2% halothane
mixed with 70% nitrogen and 28% oxygen. Body temperature
was maintained at 37.5
o
C
±
0.5
o
C during surgery, by rectal
thermistor coupled to a heating lamp.
The method for producing reversible focal ischemia has been
fully described.
6
The right common carotid artery was isolated
through a ventral midline neck incision and ligated. A 1cm
incision was made perpendicular to and bisecting a line between
the lateral canthus and the right eye and the external auditory
canal. The underlying temporalis muscle was partially excised. A
burr hole, 1 mm in diameter, was drilled 2 – 3 mm rostral to the
point of fusion of the zygomatic arch with the temporal bone.
Drilling was accompanied by a gentle drip of isotonic saline to
prevent warming of the underlying cortex. The dura over the
MCA was then cut and retracted. A #1 micro-clip (Codman) was
placed on the MCA at a site proximal to the point where it
crosses the inferior cerebral vein in the rhinal fissure. The
incisions were then closed with wound clips.
Animals were subjected to two hours of ischemia, during
which time the anesthesia was discontinued and the animals
allowed to regain consciousness. At the end of ischemia, the rats
were briefly re-anesthetized with halothane, the MCA clip
removed, and blood re-flow through the MCA visually verified.
The wound was sutured closed, and the animal permitted to
regain consciousness. Sham groups of animals were treated in
the same manner, except the micro-clip was placed on the MCA
and then removed immediately. Animals were maintained at a
rectal temperature of 37
o
C - 38
o
C for all procedures, except
where indicated otherwise in the hypothermic experiments.
Regional transfer constants (K
i
) for BBB permeation of
3
H-
sucrose were measured by a previously published method.
8,10
modified after 9
Measurements were made at different time points
after reperfusion of the right MCA. The rats were anesthetized
with pentobarbital (65 mg/kg i.p.) and, after cannulation of a
femoral artery and vein,
3
H-sucrose (NET-341) was injected
intravenously (20 µCi/100g, in 0.5 ml saline). Immediately upon
tracer injection, syringe-pump sampling of femoral arterial blood
was begun at a constant rate (0.039 ml/min
-1
) and continued for
30 min. At this point, sampling was stopped and the brain was
immediately cleared of intravascular tracer
8
by perfusing 25 ml
saline at 100 – 130 mm Hg pressure through a cannula inserted a
few minutes beforehand into the right carotid artery.
12
The rat
was decapitated, the brain removed and dissected bilaterally into
the cortex (about 180 mm
3
, representing the complete MCA
supply territory), striatum, and hippocampus. Brain samples
(weighed) and measured volumes of plasma from the arterial
sample were placed in the scintillation vials and solubilized
overnight at 37.5
o
C in 1.3 ml Soluene 350 (Packard Instr.). 10 ml
of fluor (HionicFluor) was added to all vials and the samples
were counted by liquid scintillation to determine the tracer level
in the brain parenchyma (C
paren
, dpm.g
-1
) and the time integral of
the plasma tracer level (α
1800
C
plasma
dt, dpm.s.ml
-1
). The integral
was obtained by multiplying the plasma concentration (C
plasma,
dpm.ml
-1
) by the circulation time (1800s). The transfer constant
(K
i
, mL.g
-1
.s
-1
) was calculated from the relationship: K
i
=
C
paren
/α
1800
C
plasma
dt.
9
In Experiment 1, radiotracer studies were carried out at 1, 4,
22, and 46 hours after reperfusion (or 3, 6, 24, and 48 hours after
the onset of ischemia) with five animals in each group. In an
additional group (n = 5), the radiotracer experiments were
initiated within 1.5 – 2 min after reperfusion. In this case only,
pentobarbital rather than halothane, anesthesia was induced 15
min before reperfusion for cannulations and clip removal. A
group of sham-operated rats (n = 7 total) was studied at 1, 2, 3,
5, and 24 hours after sham occlusion, with one or two rats at each
time point. No sham-operated rats were studied at 1.5 - 2
minutes.
In Experiment 2, rats underwent mild or moderate
hypothermia during the first 6 hours of reperfusion. Ten minutes
before the end of 2 hours of MCA occlusion, the rats were lightly
halothane anesthetized and were surrounded by bags of crushed
ice. This caused body temperature to drop to 32
o
C - 33
o
C at the
time that the MCA clip was removed. Rectal temperature was
maintained for 6 hours at 32
o
C - 33
o
C for the mild hypothermic
group (n=7) or 28
o
C - 29
o
C for the moderate hypothermic group
(n=7). Control animals, maintained at 37
o
C - 38
o
C, were
concurrently studied with their hypothermic peers, such that two
normothermic groups were formed (n = 8 for the mild
hypothermia controls, and n = 7 for the moderate hypothermia
controls). During the six hours, halothane was continued at 0.5 –
0.7% for the hypothermia groups only, as the control group did
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LE JOURNAL CANADIEN DES SCIENCES NEUROLOGIQUES

not require anesthesia during reperfusion. Afterwards, these
animals were permitted to recover from anesthesia and return to
normothermic conditions. Radiotracer experiments were carried
out at 46 hours post-reperfusion.
In Experiment 3, to assess edema development after two hours
of MCA occlusion, rats were sacrificed by decapitation after 4 (n
= 5), and 46 (n = 6) hours of reperfusion. Samples of right and left
cortex were dissected out and the percentage water content was
obtained by weight difference after complete oven drying. The
effect of 6 hours post-reperfusion hypothermia was also examined
in relation to edema formation. Six rats were subjected to 6 hours
of post-reperfusion moderate hypothermia (28
o
C - 29
o
C) followed
by 40 hours of normothermia (37
o
C - 38
o
C).
In Experiment 4, neocortical infarct volume was measured in
separate animals to determine the effect of hypothermia after
MCAO. Rats underwent two hours of ischemia and were then
kept either normothermic (n = 6), or immediately underwent 6
hours of mild (32
o
C - 33
o
C, n = 6) or moderate (28
o
C - 29
o
C, n
= 6) hypothermia, as above. Rats were anesthetized and
decapitated after 46 hours of reperfusion and the brains removed
and frozen. Coronal sections, 20 µm thick, were cut at -25
o
C, and
every 25th section was saved. The sections were stained with
hematoxylin and eosin, and infarct volume and edema were
obtained with an image processing system (Image-Pro II). Infarct
area of each section was traced, and the total infarct volume was
calculated by summing the infarcted area of sequential sections
and multiplying the interval thickness between sections. Edema
was calculated by comparing the increase in size of the right
hemisphere as compared to the left hemisphere.
Statistical analyses used in the transfer constant and edema
studies were the ANOVA plus Tukey’s test. A two-tailed
Student’s t test was used to compare cortical infarct volumes in
hypothermic versus control animals. A p-value of less than 0.05
was taken to indicate statistical significance. All values are
presented as ± SD.
R
ESULTS
Experiment 1
Mean regional transfer constants for the six groups of
normothermic rats are summarized in Table 1. In 7 sham-stroked
rats, radiotracer measurements were initiated at 1, 2, 3, 5, or 24
hours after the sham procedure. Each time period group
contained 1 animal, except for the 3 and 24 hour groups, which
contained 2 animals each. There were no significant differences
between the regional K
i
values for cerebral tissues on the right
side of the brain (which had undergone a complete surgical
procedure, except for MCA occlusion), and those of
corresponding territories on the left side. In the 5 experimental
groups of rats which underwent 2 hours of MCA occlusion (each
group: n = 5), the largest blood-brain barrier openings and
increases in K
i
took place in the right neocortical tissue supplied
by the occluded right MCA (Table 1). When the 30 min tracer
circulation period began 1.5 – 2 min after reperfusion, there was
evidence of an early increase in mean K
i
to greater than 10-fold
of the baseline value. This was followed by a partial recovery,
with subsequent K
i
measurements and 1 and 4 hours post-
reperfusion, which were significantly lower than the acute (1.5 –
2 min) group values, although still elevated above baseline and
above values for the contralateral, non-ischemic side. A late
opening in the BBB was then demonstrated 4 and 22 hours post-
reperfusion. This was most pronounced between 22 and 46 hours
(Figure 1).
Striatum removed from the right hemisphere of stroked rats
exhibited a slight but significant elevation in K
i
, 1.3 – 2 min post-
reperfusion (Table 1). This change was no longer present at 1 or
4 hours post-reperfusion, however, a significant increase in K
i
was demonstrated between 22 and 46 hours after reperfusion.
The dorsal hippocampus ipsilateral to the MCA occlusion
showed little change in K
i
, except at the 46 hour time point, when
a slight elevation was present. In the contralateral, non-ischemic
Table 1: Regional Transfer Constant (K
i
) in MCA Model (Experiment 1)
Right Left
(Mean ± SD) (mL.g
-1
.s
-1
x 10
6
) (Mean ± SD) (mL.g
-1
.s
-1
x 10
6
)
Variability Cortex Striatum Hippocampus Cortex Striatum Hippocampus
Group (n)
Sham (7) 1.6 ± 0.8
b
1.3 ± 0.6
b
1.7 ± 0.7 1.9 ± 0.4 1.3 ± 0.3 1.9 ± 0.3
Immediate (5) 17.3 ± 11.9
abc
3.4 ± 1.0
a
2.8 ± 1.0 2.8 ± 1.0
d
2.2 ± 0.6 3.0 ± 1.1
RP-1 Hour (5) 5.7 ± 4.1
ac
2.4 ± 1.0 2.3 ± 0.9 2.3 ± 0.4
d
1.9 ± 0.8 2.1 ± 0.4
RP-4 Hours (5) 5.9 ± 2.9
ab
2.7 ±1.9 2.0 ± 1.0 3.0 ± 1.4 2.1 ± 1.1 2.7 ± 1.1
RP-22 Hours (5) 13.1 ± 2.0
abc
5.3 ± 1.0
ab
2.4 ± 0.5
b
2.3 ± 0.7
d
1.6 ± 0.4
d
2.3 ± 0.9
RP-46 Hours(5) 59.0 ± 9.3
ac
16.5 ± 6.8
a
6.0 ± 2.6
a
2.5 ± 0.8
d
2.1 ± 0.78
d
2.7 ± 0.8
p<0.05 for ANOVA plus Tukey’s test.
Within tissues and between treatments: a – different from control (sham stroke) value; b – different from value immediately below.
Within treatments and between tissues: c – different from all other values; d – left side values different from corresponding right side values.
300
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Volume 26, No. 4 – November 1999
301
hemisphere, all regions exhibited mean K
i
values slightly higher
then the baseline, sham stroke values. However, in no instance
was this statistically significant.
Experiment 2
Table 2 shows the effect of mild or moderate hypothermia for
6 hours post-reperfusion on BBB opening caused by MCAO.
Mean K
i
measured 46 hours post-reperfusion was lower in rats
that underwent hypothermia (32
o
C - 33
o
C) compared to that of
normothermic controls. With moderate hypothermia of 28
o
C -
29
o
C, the reduction in BBB opening was more striking.
Experiment 3
Table 3 reports edema measurements based on the wet to dry
weight difference as a percentage of water per cerebral
hemisphere (ml.g
-1
x 100). In 5 rats sacrificed 4 hours post-
reperfusion, the values (mean ± SD) were 80.4 ± 0.3% and 78.5
± 0.2% for the right and left sides, respectively (p<0.001).
Furthermore, the mean % H
2
O measured in the stroked
hemisphere at 22 hours post-reperfusion was significantly higher
than that at 4 hours (p< 0.01), whereas, in the non-ischemic
hemisphere there was no significant difference between these
two time points.
Table 2: Effect of Mild and Moderate Post-Ischemic Hypothermia on Transfer Constant (K
i
) (Experiment 2)
Right Left
(Mean ± SD) (mL.g
-1
.s
-1
x 10
6
) (Mean ± SD) (mL.g
-1
.s
-1
x 10
6
)
Variability Cortex Striatum Hippocampus Cortex Striatum Hippocampus
Group (n)
Normothermia (8) 30.6 ± 9.1 5.2 ± 3.0 2.0 ± 0.5 1.2 ± 0.2 1.1 ± 0.3 1.5 ± 0.3
Hypothermia (7) 23.1 ± 9.1 4.0 ± 1.5 1.9 ± 0.7 1.3 ± 0.4 1.1 ± 0.5 1.6 ± 0.7
32-33ºC
Normothermia (7) 34.7 ± 10.9 5.0 ± 1.3 2.7 ± 1.2 2.0 ± 0.4 1.0 ± 1.0 2.3 ± 1.0
Hypothermia (7) 15.4 ± 8.8** 3.7 ± 1.7 1.8 ± 0.7 1.5 ± 0.2 1.2 ± 0.3 1.8 ± 0.4
28-29ºC
** p<0.01, student t-test.
Figure 1: Biphasic opening of the blood-brain barrier in right cerebral cortex of the rat after 2 hours of occlusion and 46 hours of reperfusion of the
right MCA. Opening of BBB indicated by increased transfer constant K
i
for blood-to-brain diffusion of
3
H-sucrose. The K
i
values for the right cortex
(ipsilateral to the MCA occlusion) and left cortex from individual animals are represented by open and shaded circles, respectively. The open square
with error bars represent the mean ±SD for each time period. ** p<0.01; *** p<0.001, significantly elevated from control values.

Post-ischemic hypothermia (28
o
C - 29
o
C) for the first 6 hours
had no ameliorative effect upon the edema measured in the
cortex 46 hours after reperfusion. The mean ( ± SD) percentage
water (ml.g
-1
x 100) for the normothermic group was 86.1 ±
0.7% for the post-ischemic right cortex and 79.8 ± 0.7% for the
contralateral left cortex, versus 85.7 ± 1.4% (right) and 79.6 ±
0.3% (left) for that of the hypothermic group.
Experiment 4
Despite the failure of moderate post-ischemic hypothermia to
reduce the accumulation of edema in hypothermia (28
o
C - 29
o
C)
for the first 6 hours of the 46 hour reperfusion period, this resulted
in a mean cortical infarct volume of 122 ± 57 mm
3
(n = 6) (Figure
2), which was significantly lower than that of the normothermic
control group (175 ± 22 mm
3
, n = 6) (p < 0.05) (Table 4). (Table
4). Animals receiving 6 hours of mild hypothermic reperfusion
(32
o
C - 33
o
C) had a total infarct volume of 149 ± 39 mm
3
(n = 6)
at 46 hours, which was less than that of the control group, but not
significantly so. The reduction in the size of injury relates to
smaller volumes of infarction rather than differences in the
amount of swelling or edema, confirming Experiment 3.
D
ISCUSSION
In these transient ischemic experiments, the widest BBB
openings and increases in K
i
were seen in the post-ischemic right
cerebral cortex, which was dissected to include both the core and
edge of the region perfused by the MCA. The opening was
clearly biphasic, characterized by an initial 10-fold augmentation
in K
i
during the first half hour of reperfusion, followed by partial
closing, and then a delayed, but progressive, opening between 22
and 46 hours post-reperfusion. Moderate hypothermia during
ischemia dramatically reduced infarction and edema, as well as
preventing BBB opening, but also had partial effects on infarct
size when instituted during the post-ischemic period following
normothermic ischemia.
11
In these studies, we have
demonstrated that postischemic moderate hypothermia affects
not only the size of the infarct, it does so in tandem with
reductions in the opening of the BBB, not by simply reducing the
amount of vasogenic edema, but possibly by interfering with the
post-ischemic inflammatory response.
This profile of BBB injury shows similarities to findings based
on the assessment of Evan’s Blue dye extravasation in the cat.
13
Following one hour of temporary MCA occlusion, dye injected i.v.
early in reperfusion caused staining of brain parenchyma. This was
followed by a refractory period, and then a delayed opening, which
was visible in cats sacrificed five hours or three days post-stroke.
The initial acute opening has been described as a ‘hemodynamic’
BBB opening.
4,13
Because of acidosis, loss of autoregulation, and
vasodilation of the cerebral vasculature, reperfusion results in
excessive blood flow or ‘luxury perfusion’. High intraluminal
blood pressure in the cerebral microvasculature has been shown to
induce abnormal pinocytotic transport across endothelial cells, and
opening of interendothelial tight junctions.
14,15
A significant role of
arterial pressure in the degree of post-reperfusion opening after
Table 3: Percentage of Water in MCA Model (Experiment 3)
Variability Right Cortex Left Cortex
Group (n) % of Water (Mean ± SD) % of Water (Mean ± SD)
Normothermia
4 hours post-reperfusion (5) 80.4 ± 0.3
a
78.5 ± 0.2
22 hours post-reperfusion (5) 83.7 ± 0.6
ab
78.8 ± 0.1
46 hours post-reperfusion (6) 86.1 ± 0.7
ab
79.8 ± 0.7
Moderate Hypothermia (6) 85.7 ± 1.4
ab
79.6 ± 0.3
p < 0.01 for ANOVA plus Tukey’s test.
a – right side values different from corresponding left side values; b – different from 4 hr post-reperfusion value.
Figure 2: Volume of neocortical infarction for each rat following 2
hours of normothermic transient MCA occlusion and 46 hours of
reperfusion, in the first 6 hours of which animals were maintained at
either 37
o
C-38
o
C (normothermia), 32
o
C-33
o
C (mild hypothermia), or
28
o
C-29
o
C (moderate hypothermia). The mean edema and infarct sizes
are displayed ±SD (the asterisk denotes significantly less injury
(p<0.05). The n value for each group is indicated in brackets.
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302

MCAO has been demonstrated.
16
After 3 hours of MCAO and 30
min re-circulation, BBB opening to Evan’s Blue dye was greatly
augmented in rats that had been rendered hypertensive with
phenylephrine during the reperfusion period. The magnitude of
acute hyperemic BBB opening, and of modulating factors, such as
blood pressure, would appear to be an important consideration in
utilizing and interpreting the MCAO model. For example, factors
such as hypertension, which augment post-reperfusion opening,
and the ensuing homeostatic changes might thereby indirectly
influence subsequent neuropathological events. On the other hand,
one might anticipate that the degree of early post-ischemic BBB
opening could have a positive impact on the efficacy of
experimental drugs when the chemical nature of these compounds
limits their ability to cross the normal BBB. For instance, with the
competitive AMPA antagonist NBQX, which penetrates the blood-
brain barrier, effective concentrations of the drug are achieved
following transient focal ischemia, perhaps as a consequence of
the opening of the barrier at the time the drug is circulating.
17
In this study, partial recovery from the acute opening of the
BBB was evidenced by the fact that K
i
values at one or four
hours post-reperfusion were significantly lower than those
measured acutely, but were higher than baseline values, or values
for contralateral non-ischemic cortex. The second part of the
biphasic opening was then indicated by the significant elevation
of K
i
at 22 hours post-reperfusion as compared to that at four
hours, with an even more dramatic upward increment taking
place between 22 and 46 hours. Accounting for this time delay
would seem important in any proposal on the cause(s) of BBB
opening, which presumably differs from that underlying the
acute post-ischemic opening. In fact, the delayed BBB opening
to
3
H-sucrose is consistent with the published observations that
between 24 and 48 hours after transient MCAO, there occurs a
rapid evolution of delayed edema, which peaks within this time
period, and that infiltration of polymorphonuclear cells follows a
similar time course.
18
Vascular endothelial leakiness was
proposed to result from the release of lipid inflammatory
mediators through the interaction of injured tissue with
infiltrating leukocytes and aggregating platelets.
19
Among the
possible mediators of BBB dysfunction and formation of
vasogenic edema, proteases, bradykinin, histamine, and
eicosanoid products of arachidonic acid metabolism and free
radicals have been strongly implicated.
20
It is well documented that neuronal injury is reduced by
induction of hypothermia during ischemia, or even by its
induction during the post-ischemic reperfusion period.
21
The
experiments in the present study quantitate for the first time a
protective effect of post-ischemic hypothermia on the delayed
BBB injury that follows temporary MCAO. Mean K
i
values after
46 hours of reperfusion were more than 50% lower in rats that
underwent cooling to 29
o
C for 6 hours. Although a light degree
of halothane anesthesia in the cooled rats may have contributed
a protective effect, efficacy of lowered brain temperature per se
was suggested by the fact that 28
o
C - 29
o
C was more protective
than 32
o
C - 33
o
C. Separate experiments showed, however, that
the 6 hours of hypothermic treatment at 28
o
C - 29
o
C did not
reduce the amount of edema present at 46 hours post-reperfusion,
even though this treatment appeared to favourably affect both
BBB damage and infarct volume.
Post-ischemic hypothermic protective mechanisms may be
related to a slight attenuation in the reperfusion hyperemia.
During reperfusion, hypothermia may reduce leukotrienes,
22
improve glucose utilization and blood flow,
23
and slow free
radical reactions and the propagation of lipid peroxidation
cascades. This could prevent the leakage of proteins and the
accumulation of extracellular fluid
24
and inhibit the biosynthesis,
release and uptake of neurotransmitters, such as glutamate and
dopamine.
25
There are two massive glutamate release points in
the first 4 hour period of reperfusion following MCA/CCA
occlusion.
26
The quantal release of glutamate has been recently
correlated with the size of neocortical infarction in focal
ischemia
27
and may result in endothelial cell damage in the BBB.
The blockade on the non-NMDA glutamate receptors attenuates
brain damage.
17
Therefore, any reduction of glutamate
concentration in the extracellular space by delayed hypothermia
may protect against brain damage.
In conclusion, following transient focal ischemia,
measurements of K
i
for BBB permeation of
3
H-sucrose have
demonstrated both an acute opening, likely hemodynamic in
nature, and a delayed opening of the ipsilateral cortex MCAO.
Clearly, hypothermia has potent effects on BBB opening
28
and
does reduce infarct size, but through mechanisms other than
reducing edema. The quantitative power of K
i
measurements
should facilitate the exploration of drug or other treatments to
offset the disadvantages of reperfusion therapy (BBB opening
and edema) and facilitate its benefits. The understanding of the
interplay between microvascular damage, edema, inflammation,
and neuronal death hours after thromboembolic stroke is critical
to the development of successful stroke therapies.
Table 4 : Effects of infarct volume and edema formation following post-ischemic hypothermia in MCA Model (Experiment 4)
Variability Edema Infarction Total Infarct Volume
Group (n) (mm
3
) (Mean ± SD) (mm
3
) (Mean ± SD) (mm
3
) (Mean ± SD)
Normothermia (6) 58 ± 17 117 ± 13 175 ± 23
Mild Hypothermia (6) 67 ± 23 83 ± 31* 149 ± 39
Moderate Hypothermia (6) 43 ± 26 79 ± 33* 122 ± 57
* p< 0.05, between treatments for ANOVA test.
Volume 26, No. 4 – November 1999
303
LE JOURNAL CANADIEN DES SCIENCES NEUROLOGIQUES

ACKNOWLEDGEMENTS:
Supported, in part, by grants from the Heart and Stroke Foundation of
Canada. Alastair Buchan is a Senior Scholar of the Alberta Heritage
Medical Research Foundation.
REFERENCES
1. Rapoport SI. Sites and function of the blood-brain barrier. In:
Rapoport SI, ed. Blood-Brain Barrier in Physiology and
Medicine. New York: Raven Press, 1976: 43-86.
2. Betz AL. Oxygen free radicals and the brain microvasculature. In:
Pardridge WM, ed. The Blood-Brain Barrier. Cellular and
Molecular Biology. New York: Raven Press, 1993: 303-321.
3. Betz AL, Keep RF, Beer ME, Ren X-d. Blood-brain barrier
permeability and brain content of sodium, potassium and chloride
during focal ischemia. J Cereb Blood Flow Metab 1994; 14: 29-
37.
4. Klatzo I. Concept of ischemic injury associated with brain edema.
In: Inaba Y, Klatzo I, Spatz M, eds. Brain Edema. Tokyo:
Springer, 1984: 1-5.
5. Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W. Focal
brain ischemia in the rat: methods for reproducible neocortical
infarction using tandem occlusion of the distal middle cerebral
and ipsilateral common carotid arteries. J Cereb Blood Flow
Metab 1988; 8: 474-485.
6. Buchan AM, Xue D, Slivka A. A new model of temporary focal
neocortical ischemia in the rat. Stroke 1992; 23: 273-279.
7. Kaplan B, Brint S, Tanabe J, Jacewicz M, Wang X-J, Pulsinelli W.
Temporal thresholds for neocortical infarction in rats subjected to
reversible focal cerebral ischemia. Stroke 1991; 22: 1032-1039.
8. Preston E, Allen M, Haas N. A modified method for measurement of
radiotracer permeation across the rat blood-brain barrier: the
problem of correcting brain uptake for intravascular tracer. J
Neurosci Meth 1983; 9:45-55.
9. Ohno K, Pettigrew KD, Rapoport SI. Lower limits of
cerebrovascular permeability to nonelectrocytes in the conscious
rat. Am J Physiol 1978; 235: H299-H307.
10. Preston E, Haas N. Defining the lower limits of blood-brain barrier
permeability: factors affecting the magnitude and interpretation
of permeability-area products. J Neurosci Res 1986; 6: 709-716.
11. Xue D, Huang ZG, Smith KE, Buchan AM. Immediate or delayed
mild hypothermia prevents focal cerebral infarction. Brain Res
1992; 587: 66-72.
12. Ishimaru S, Hossman KA. Relationship between cerebral blood
flow and blood-brain barrier permeability of sodium and albumin
in cerebral infarcts of rats. Acta Neurochir 1990; 51(S): 216-219.
13. Kuroiwa T, Ting P, Klatzo I. The biphasic opening of the blood-
brain barrier to proteins following temporary middle cerebral
artery occlusion. Acta Neuropathol 1985; 68: 122-129.
14. Westergaard E, van Deurs B, Brondsted HE. Increased vesicular
transfer of horseradish peroxidase across cerebral endothelium,
evoked by acute hypertension. Acta Neuropathol 1977; 37: 141-152.
15. Nagy Z, Mathieson G, Huttner I. Blood-brain barrier opening to
horseradish peroxidase in acute arterial hypertension. Acta
Neuropathol 1979; 48: 45-53.
16. Cole DJ, Matsumura JS, Drummond JC, Schultz RL, Wong MH.
Time- and pressure-dependent changes in blood-brain barrier
permeability after temporary middle cerebral artery occlusion in
rats. Acta Neuropathol 1991; 82: 266-273.
17. Buchan AM, Xue D, Huang ZG, Smith KE, Lesiuk H. Delayed
AMPA receptor blockade reduces cerebral infarction induced by
focal ischemia. NeuroReport 1991; 2: 473-476.
18. Hsu CY, Liu TH, Xu J, et al. Arachodonic acid and its metabolites
in cerebral ischemia. Ann NY Acad Sci 1989; 559: 282-295.
19. Hsu CY, Liu TH, Xu J, Hogan EL, Chao J. Lipid inflammatory
mediators in ischemic brain edema and injury. In: Bazan NG ed.
Lipid Inflammatory Mediators in Ischemic Brain Damage and
Experimental Epilepsy. New Trends Lipid Mediators Research.
Basel: Karger, 1990: 85-112.
20. Wahl M, Unterberg A, Baethmann A, Schilling L. Mediators of
blood-brain barrier dysfunction and formation of vasogenic brain
edema. J Cereb Blood Flow Metab 1988; 8: 621-634.
21. Ginsberg MD, Sternau LL, Globus MYT, Dietrich WD, Busto R.
Therapeutic modulation of brain temperature: relevance to
ischemic brain injury. Cerebrovasc Brain Metab Rev 1992; 4:
189-225.
22. Dempsey RJ, Combs DJ, Edwards MM. Moderate hypothermia
reduces post-ischemic edema development and leukotriene
production. Surgery 1987; 21: 177-181.
23. Busto R, Dietrich WD, Globus MY, Ginsberg MD. Postischemic
hypothermia inhibits CA1 hippocampal ischemic neuronal injury.
Neurosci Lett 1989a; 101: 299-304.
24. Dietrich WD, Halley M, Valdes I, Busto R. Interrelationship
between increased vascular permeability and acute neuronal
damage following temperature controlled brain ischemia in rats.
Acta Neuropathol 1991; 81: 615-625.
25. Busto R, Globus MYT, Dietrich WD, et al. Effects of mild
hypothermia on ischemia-induced release of neurotransmitter and
free fatty acids in rat brain. Stroke 1989b; 20: 904-910.
26. Osuga H, Hakim AM. The changes in extracellular glutamate
concentration during focal ischemia in rat. Can J Neurol Sci
1992; 19: 301-302.
27. Butcher SP, Bullock R, Graham DI, McCulloch J. Correlation
between amino acid release and neuropathologic outcome in rat
brain following middle cerebral artery occlusion. Stroke 1990;
21: 1727-1733.
28. Preston E, Sutherland G, Finsten A. Three openings of the blood-
brain barrier produced by forebrain ischemia in the rat. Neurosci
Lett 1992; 149: 75-78.
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