(Stroke. 1998;29:683-689.)
© 1998 American Heart Association, Inc.
Effect of Vasospasm on Heme Oxygenases in a Rat Model of Subarachnoid Hemorrhage
Minoru Kuroki, MD;
Kenji Kanamaru, MD, DMSc;
Hidenori Suzuki, MD;
Shiro Waga, MD, DMSc;
Reiji Semba, MD, PhD
From the Departments of Neurosurgery (M.K., K.K., H.S., S.W.) and Anatomy
II (R.S.), Mie University School of Medicine, Mie, Japan.
Correspondence to: Kenji Kanamaru, MD, DMSc, Department of Neurosurgery, Mie University School of Medicine, Tsu, Mie 514, Japan. E-mail kanamaru{at}clin.medic.mie-u.ac.jp
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Abstract
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Background and
Purpose—Subarachnoid hemorrhage (SAH)-induced
heme oxygenase-1 (HO-1) in glia throughout the rat brain
without affecting heme oxygenase-2 (HO-2). However, the
relationship between cerebral vasospasm and the expression of heme
oxygenases after SAH is thus far unknown. The purpose of
the present study was to clarify the effect of vasospasm on the
expression of heme oxygenases in a rat model of
SAH.
Methods—Endothelin, hemolysate, hemolysate saturated with carbon
monoxide (CO-hemolysate), and saline were injected into the cisterna
magna of adult rats. Angiography was repeated before each injection and
15 and 60 minutes and 24 hours after each injection.
Immunocytochemistry for HO-1, HO-2, and glial fibrillary acidic protein
(GFAP) was performed 24 hours after the injection.
Results—A significant vasospasm occurred in the basilar artery
after the injection of endothelin, hemolysate, and CO-hemolysate. The
degree of vasospasm was most prominent 15 minutes after each injection.
There was no significant difference in the degree of vasospasm among
injections. The HO-1 was induced exclusively in the glial cells
throughout the brain after injection of hemolysate and CO-hemolysate;
however, it was not induced by endothelin and saline. In the dentate
gyrus of the hippocampus and the molecular layer of the cerebellum, the
HO-1-positive cells were also stained for GFAP, suggesting astrocytic
glial cells. On the other hand, HO-2 immunoreactivity was abundant in
neurons and was not affected by endothelin, hemolysate, CO-hemolysate,
or saline.
Conclusions—It is suggested that heme per se, rather than
ischemia induced by vasospasm, plays a pivotal role in the
expression of HO-1 in this rat model.
Key Words: subarachnoid hemorrhage • vasospasm • heme oxygenase • endothelins
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Introduction
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Despite the fact that
subarachnoid hemorrhage (SAH) causes vasospasm and
induces stress proteins such as HSP70 and heme oxygenase-1
(HO-1),1 2 3 4 it is still unknown whether the
stress proteins are caused by the vasospasm or the SAH itself. Free
radical generation and lipid peroxidation may be very important for the
occurrence of vasospasm after SAH.5 6 7 8 Heme
oxygenase catalyzes the rate-limiting step in heme
degradation, yielding iron, biliverdin, and carbon monoxide (CO);
biliverdin is subsequently reduced enzymatically to bilirubin by
biliverdin reductase.9 Both bilirubin and CO are
thought to have important physiological functions,
bilirubin as an antioxidant10 and CO as a
messenger molecule.11
HO exists as two isozymes, namely, HO-1 and HO-2. HO-2 is the
constitutive form, and under normal conditions it is by far the
predominant isozyme in the brain, where it is expressed in the
cerebellum, forebrain, brain stem, and
diencephalon.12 HO-1 under normal conditions is
detectable in selective neuronal populations of the hippocampus and in
the 
-motor neurons in the spinal cord, the red nucleus, and dorsal
raphe.12 However, HO-1 is induced in response to
stress inducers such as oxidative stress, cadmium, ultraviolet light,
phorbol ester, and hypoxia.9 To date,
direct demonstration of the relationship between vasospasm and heme
oxygenase after
SAH has not been researched. In the present study, we
investigated the effects of vasospasm on heme oxygenases in
a rat model of SAH.
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Materials and Methods
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All protocols were evaluated and approved by the Animal Ethics
Review Committee of the Mie University School of Medicine. The animals
were cared for in accordance with the guideline for animal experiments
at the Mie University School of Medicine.
Study Protocol
Male Sprague-Dawley rats weighing 350 to 500 g (SLC) were
anesthetized by intraperitoneal injection
of chloral hydrate (400 mg/kg), intubated with a 16- or 18-gauge
elastic catheter, and allowed to breathe spontaneously. The rectal
temperature was kept at approximately 37°C with an electric heating
pad, and end-tidal CO2 was monitored (POET 601,
Criticare System Inc). Anesthesia was maintained by
repeated injections of chloral hydrate, if needed. After cannulation of
both radial arteries with 22-gauge catheters, 0.3 mL autologous
arterial blood was withdrawn, and the same volume of saline
was injected in hemolysate and CO-saturated hemolysate (CO-hemolysate)
groups. Both catheters were connected with a three-way stopcock to a
pressure transducer (MK 12030 US, Baxter) to measure blood pressure.
Vertebral angiography was performed by manual injection of a contrast
medium (0.3 mL Iomeron 400, Eisai) into both radial arteries
simultaneously. The basilar artery diameter was determined
at three different points: 0.5 mm above the junction of the
vertebral arteries and halfway and just below the bifurcation of the
posterior cerebral arteries. During the angiography, exposure factors
were kept constant, and a radiopaque control standard was used for
correction to constant magnification. An experienced person who was
unaware of the treatment groups measured the diameter of the basilar
artery three times with a calibrated optical micrometer
(scale loupe No. 1983, PEAK).
After angiography, animals were placed in a head holder with the head
at 30° of flexion. A midline skin incision was made from the middle
of calvarium to the lower cervical spine. The occipital bone was
cleared of muscular attachments by sharp dissection. Under a surgical
microscope, the atlanto-occipital membrane was dissected and punctured
for the injection of each material. The animals were randomly assigned
into five groups: a sham-operation group (n=3), a saline group (n=3),
an endothelin group into which 1.2x10-10 mol/kg
endothelin (Sigma) was injected (n=7), a hemolysate group (n=6), and a
CO-hemolysate group (n=6). After the injection of each material,
angiography was repeated at 15 and 60 minutes. The catheters were then
removed, and the wounds were closed. After 24 hours the animals were
anesthetized again and a third angiography was performed in the
same manner as described above. During the course of observation, the
animals were allowed access to food and water ad libitum.
Immunocytochemistry for Heme Oxygenases
The animals in the sham-operation (n=3), saline (n=3),
endothelin (n=6), hemolysate (n=6), and CO-hemolysate (n=6) groups were
killed 24 hours after the operation. The rats were anesthetized
with intraperitoneal chloral hydrate (600 mg/kg)
and perfused with the use of a cardiac catheter with 200 mL normal
saline followed by 500 mL 4% paraformaldehyde in 0.1
mol/L phosphate buffer saline (PBS, pH, 7.4). The brains were postfixed
in 4% paraformaldehyde for 3 to 6 hours and incubated
in 10%, 20%, and 30% sucrose in 0.1 mol/L PBS by steps, each for 8
hours at 48°C. Sagittal sections were cut into 50 mm thicknesses
with a sliding microtome and placed in PBS containing 0.02% sodium
azide. Tissue sections were washed twice with PBS for 5 minutes.
Endogenous peroxidase was blocked by incubation with 0.3%
hydrogen peroxide in PBS for 60 minutes, and nonspecific binding of
antibodies was prevented by preincubation of sections in 10% normal
goat serum. Sections were then incubated for 16 hours with antibodies
against HO-1 (OSA 100, StressGen Biotechnologies) at a dilution of
1:1000 or against HO-213 at a concentration of
1.2 mg/mL in PBS containing 10% normal goat serum. Alternate control
sections were incubated without primary antibody and demonstrated no
staining. After being washed in PBS, sections were incubated with
biotinylated rabbit IgG antibodies (1:200) and freshly prepared ABC
solution (1:50; Vectastain; Vector Laboratories), each for 1 hour.
Peroxidase activity was revealed by incubation with
3,3'-diaminobenzidine tetrahydrochloride (Dojindo Chemical Institute)
with 0.01% H2O2, and the
sections were dehydrated, cleared, and mounted with Entellan
(Merck).
Immunofluorescent Double Labeling for Astrocyte
The immunogold-silver staining method for HO-1 followed by FITC
double staining for GFAP was used. Sections were washed in PBS, coated
with Logol's iodine solution for 10 minutes, and rinsed thoroughly.
All traces of iodine were then removed with 2.5% sodium thiosulfate,
and the sections were incubated with 1% skim milk for 10 minutes to
block nonspecific background staining. Sections were incubated with
antibodies against HO-1 for 16 hours, washed with PBS, and left in
gold-labeled goat anti-rabbit IgG for 3 hours. After being washed with
PBS and 0.2 mol/L phosphate buffer (pH 7.2) containing 0.1% gum
arabic, the sections were developed with the silver enhancement kit
(Amersham) for 20 minutes. Sections were then washed and incubated with
rabbit polyclonal anti-GFAP for 24 hours at 48°C. Alternate control
sections were incubated without anti-GFAP and demonstrated no staining.
After being washed in PBS, the sections were incubated with
FITC-conjugated goat anti-rabbit IgG antibody for 2 hours. Sections
were placed on a coverslip in a mixture of glycerol/PBS (4:1), and
fluorescence microscopy was performed with use of a microscope
with a specific filter for FITC.
Statistical Analysis
Data were expressed as mean±SD. Comparisons within groups and
intergroup comparisons were made with the use of ANOVA. The level of
significance of all tests of comparison was P<.05.
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Results
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Neurological Deficit and Physiological Parameters
No rats developed neurological deficits. Comparisons of mean
values for body weight, mean arterial blood pressure, heart
rate, and end-tidal CO2 measured before and at 15
and 60 minutes and 24 hours after injection revealed there were no
significant differences in the groups (Table 1
).
Angiographic Vasospasm
There was no significant vasospasm in the saline group. On the
other hand, a significant vasospasm occurred in the basilar artery
after intracisternal injections of endothelin, hemolysate, and
CO-hemolysate (Fig 1). Vasospasm was most
prominent at 15 minutes after each injection. At 60 minutes after
injection, a significant vasospasm was noted in the hemolysate and
CO-hemolysate groups. The basilar artery diameter returned to
preinjection level after 24 hours.
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Figure 1. The percentage of change from the baseline in the
diameter of the basilar artery in the saline, endothelin, hemolysate,
and CO-hemolysate injection groups. Percentages indicate mean±SD
values in each group. pre indicates pretreatment; m, minutes after each
treatment; and h, hours after each treatment. *P<0.05,
**P<0.05.
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Expression of HO-1
HO-1 immunoreactivity was detectable in the brains of
sham-operated and saline-injected control rats (Fig 2A through 2C). HO-1-positive cells were
noted in the hippocampal neurons, especially in the hilum of the
dentate gyrus and hypothalamus. Occasionally, solitary HO-1
immunoreactive glial cells were observed in the striatum or cerebral
cortex. In addition, the ependyma was slightly stained throughout the
ventricles. After the injection of endothelin, HO-1 immunoreactivity
was similar to that seen in the sham-operated and saline-injected
groups (Fig 2E through 2G).
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Figure 2. HO-1 immunocytochemistry after administration of
saline (panels A, B, C, and D) and endothelin (E, F, G, and H). Note
the absence of HO-1 staining in the cortex (A and E) and cerebellum (C
and G) and the sparse neuronal staining in the hippocampus (arrows in B
and F). HO-2 in the cerebellum was abundant but did not change after
injections of saline (D) and endothelin (H). Bar=100 µm.
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Marked induction of HO-1 immunoreactivity was noted throughout most
regions of the brain following injection of hemolysate (Fig 3A through 3C) and CO-hemolysate (Fig 3E
through 3G). A significant increase in the numbers of HO-1-positive
cells occurred in most brain regions of the animals that received
hemolysate and CO-hemolysate (Figs 2
and 3). The increased HO-1
immunoreactivity was localized in small cells diffusely distributed
throughout the neocortex, hippocampus, and hindbrain (Fig 3).
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Figure 3. Heme oxygenase immunoreactivity after
administration of hemolysate (panels A, B, C, and D) and CO-hemolysate
(E, F, G, and H). Note remarkable induction of HO-1 protein in the
cortex (A and E), hippocampus (B and F), and cerebellum (C and G). HO-2
immunoreactivity did not change after injections of hemolysate (D) and
CO-hemolysate (H). Bar=100 µm.
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Expression of HO-2
HO-2 immunoreactivity was noted in neurons throughout the brain.
HO-2 expression in the hemolysate and CO-hemolysate groups was similar
to that observed in the sham-operated and saline-injected animals (Figs 2D and 2H, 3D and 3H). There was no increase or decrease of HO-2
immunostaining detected in the animals in any of the
groups in this study.
Double Labeling for HO-1 and GFAP
Double-labeling immunocytochemical studies revealed that HO-1 was
induced primarily in GFAP immunoreactive astrocytes in the hippocampus
(Fig 4A and 4B) and cerebellum (Fig 4C and 4D). In the cerebellum, HO-1 immunoreactivity was observed in the
Bergmann glia cells, which extend long processes into the molecular
layer (Fig 4C and 4D).
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Figure 4. Double-labeling staining for HO-1 (panels A and C)
and GFAP (B and D). Sections from the hippocampus (A and B) and
cerebellum (C and D) were obtained from a subject receiving hemolysate
injection. The HO-1 immunoreative cells in the hippocampus (arrows in
A) appear to be GFAP-positive astrocytes (arrows in B). Note that the
HO-1 immunoreactive cells in the cerebellum (arrows in C) appear to be
mostly GFAP-positive Bergmann astrocytes (arrows in D). Bar=100
µm.
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Discussion
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The major findings of the present study were
as follows: (1) significant vasospasm occurred in the basilar artery
after injection of endothelin, hemolysate, and CO-hemolysate; (2) HO-1
was induced exclusively in glial cells throughout the brain after
injection of hemolysate and CO-hemolysate but was not induced by
endothelin or saline; and (3) no increase or decrease of HO-2
immunostaining was detected in animals in any of the
groups.
This is the first study to demonstrate angiographic vasospasm and
evaluate the effect of vasospasm on heme oxygenase
expression in a rat model. Endothelin, hemolysate, and CO-hemolysate
all induced a significant vasospasm to the same degree. Reportedly, a
40% decrease of cerebral blood flow was demonstrated in the same rat
model of SAH.14 It was inferred that if cerebral
ischemia plays a major role in the expression of HO-1,
induction of HO-1 would be observed in these three groups. However,
HO-1 was not induced after the injection of endothelin in spite of
vasospasm. It was demonstrated that injections of the protein BSA into
the cisterna magna also did not induce HO-1.2
Hence, neither a nonspecific protein nor a compound that produces
vasospasm induce HO-1 throughout the brain. Therefore, cerebral
ischemia after the injection of endothelin, hemolysate, and
CO-hemolysate might not be a major causative factor for the expression
of HO-1 in this model. Rather, heme per se might play a pivotal role in
the expression of HO-1. The large amount of heme in blood is likely
taken up into cells where it induces HO-1 and in turn is metabolized by
HO-1.2 It was reported that oxyhemoglobin may be
the causative factor of vasospasm in SAH. In the CO-hemolysate group,
oxyhemoglobin was eliminated by saturating the hemolysate with CO gas.
However, the hemolysate without oxyhemoglobin was as efficient as
hemolysate with oxyhemoglobin in inducing acute vasospasm and HO-1.
Therefore, oxyhemoglobin does not seem to be essential for the
induction of acute vasospasm and HSP; CO-hemoglobin is as efficient as
oxyhemoglobin. Because lipid peroxydation takes place in a
subarachnoid clot,8 both nonenzymatic and
enzymatic lipid peroxidation metabolites should be inducers of
HO-1.15 HO-1 might be induced by the peroxydated
lipid formed in the hemolysate. However, this model does not produce
chronic cerebral vasospasm, and in this sense it has significant
limitations in the study of the clinical entity of SAH. Thus, the
expression of HO-1 in chronic vasospasm remains to be investigated.
It has been shown that SAH induces HO-1 in Bergmann glia in the
molecular layer but not in cells in other layers, and there is diffuse
induction in the glia in both cerebellar hemispheres and
vermis.2 4 Hence it was postulated that the HO-1
induction in the cerebellum might reflect direct injury by blood
products due to local release of large amounts of catalytically
active iron and resulting oxidative injury.4
Since heme oxygenases are the rate-limiting step in the
metabolism of heme to bilirubin,9
increases in their activity should increase the rate of degradation of
heme, thus increasing the formation of bilirubin, which may in turn
play a protective role as an antioxidant.10
Increased degradation of heme also increases the release of free iron
from heme and allows the cell to begin the process of iron
sequestration through ferritin.16 Degradation of
heme produces what may be a physiological
regulation of cGMP and plays a role in the regulation of cerebral blood
flow through smooth muscle relaxation and
vasodilatation.11 17 In rabbit and canine basilar
arteries, however, it has been suggested that a direct effect of CO was
not likely and that a reduction in oxygen delivery due to changes in
the oxyhemoglobin dissociation curve may be responsible for increases
in cerebral blood flow.18 The effects of CO on
cerebral arterioles remains to be
clarified.18
We are unaware of any previous demonstration of angiographic vasospasm
after intracisternal injection of endothelin in
rats.19 A number of points favor a role for
endothelins as mediators of vasospasm, namely: (1) endothelins are
extremely potent constrictors of human cerebral
vessels20 and induce chronic vasospasm in
dogs21 ; (2) endothelin A receptor
antagonist FR139317 has been shown to reduce vasospasm in a
two-hemorrhage model of SAH22 ; (3)
production of endothelin is stimulated by various vasoactive
substances present in the blood, including arginine vasopressin,
angiotensin II, and thrombin19
(excessive local production of these substances is reported
after SAH23 24 ); and (4) excessive concentrations
of oxyhemoglobin may increase production of
endothelin25 26 (significant increases of
endothelin in the cerebrospinal fluid coincides with the appearance of
clinically relevant vasospasm27 ).
In conclusion, intracisternal injection of endothelin did not induce
HO-1. Heme per se, rather than ischemia induced by vasospasm,
therefore plays a pivotal role in the expression of HO-1 in this rat
model.
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Selected Abbreviations and Acronyms
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| CO-hemolysate |
= |
hemolysate saturated with carbon dioxide |
| GFAP |
= |
glial fibrillary acidic protein |
| HO-1 |
= |
heme oxygenase-1 |
| HO-2 |
= |
heme oxygenase-2 |
| SAH |
= |
subarachnoid hemorrhage |
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Acknowledgments
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This work was supported by a grant-in-aid for Scientific
Research (C) from the Ministry of Education, Science, Sports and
Culture and a grant-in-aid (1996) from the Mie Medical Research
Foundation (Dr Kanamaru).
Received September 2, 1997;
revision received December 5, 1997;
accepted December 15, 1997.
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Editorial Comment
Frank R. Sharp, MD, Guest Editor
Departments
of Neurology and Neurosurgery,
University of California at San Francisco,
VA Medical Center,
San Francisco, California
|
Introduction
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This study demonstrates basilar artery vasospasm following
cisterna magna injections of endothelin, red cell hemolysate, and
CO-hemolysate. This vasospasm is associated with induction of HO-1 in
microglia throughout the brain following the hemolysate but not the
endothelin injections. Because the vasospasm produced by endothelin was
similar to that produced by hemolysate injections, the diffuse HO-1
induction cannot be accounted for by ischemia produced by
vasospasm. The authors suggest that the heme in hemoglobin might be the
factor responsible for HO-1 induction after subarachnoid
injections of hemolysate. This conclusion agrees with our recent
studies1 2 3 4 showing that whole blood and lysed blood
injections into the cisterna magna of rats induce HO-1 in microglia
throughout the entire rat brain.
We have since tried to confirm that heme is the compound likely to
account for HO-1 induction after experimental subarachnoid
hemorrhage. Following subarachnoid injections of
purified hemoglobin, the hemoglobin is rapidly distributed within a few
hours in structures surrounding the ventricles and in
cortex.4 Shortly after this, HO-1 induction is detected in
microglia at the outer margins of cortex and in regions adjacent to the
ventricles. By 24 hours after cisternal injections of pure hemoglobin,
HO-1 is induced in microglia throughout the entire brain—including all
of the cortex, striatum, hippocampus, thalamus, hypothalamus, brain
stem, and cerebellum.4 We have postulated4
that heme from hemoglobin is selectively taken up into microglia
throughout the entire brain, where it induces the HO-1 gene by actions
on a heme- or iron-sensitive element in the HO-1
promoter.4 These results suggest that the microglia have
specialized systems for taking up the heme following
subarachnoid hemorrhages and for metabolizing the heme
to iron, biliverdin, and carbon monoxide.
An interesting finding not mentioned in the present study is that
subarachnoid injections of lysed blood also produce focal
regions of stress gene induction—including induction of HSP70 and
HO-1.2 3 These focal regions vary in size and location and
are produced by single injections of lysed blood but not by single
injections of pure hemoglobin.1 2 3 4 We have
speculated2 3 that these focal regions represent
regions of ischemic injury produced by vasospasm resulting from
the lysed blood injec-tions. We propose that the focal regions of
stress gene expression can be used to assess the efficacy of
drugs that might be useful in the treatment of subarachnoid
hemorrhage.
|
Selected Abbreviations and Acronyms
|
|---|
| CO-hemolysate |
= |
hemolysate saturated with carbon dioxide |
| GFAP |
= |
glial fibrillary acidic protein |
| HO-1 |
= |
heme oxygenase-1 |
| HO-2 |
= |
heme oxygenase-2 |
| SAH |
= |
subarachnoid hemorrhage |
|
Received September 2, 1997;
revision received December 5, 1997;
accepted December 15, 1997.
|
References
|
|---|
1.
Matz P, Turner C, Panter S, Weinstein PR, Sharp FR.
Heme oxygenase induction in glia throughout brain
following experimental subarachnoid hemorrhage in the
rat. Mol Brain Res.. 1996;713:211–222.
2.
Matz P, Weinstein P, States B, Honkaneimi J, Sharp FR.
Subarachnoid injections of lysed blood induce the hsp70
stress gene and produce DNA fragmentation in focal areas of rat
brain. Stroke. 1996;27:504–513.
3.
Matz P, Massa SM, Weinstein PR, Turner C, Panter S,
Weinstein PR, Sharp FR. Focal hyperexpression of
hemeoxygenase-1 protein and messenger RNA in rat brain
caused by cellular stress following subarachnoid injections of
lysed blood. J Neurosurg.. 1996;85:892–900.
4.
Turner CP, Bergeron M, Matz P, Zegna A, Noble LJ, Panter
SS, Sharp FR. Heme oxygenase-1 (HO-1) is induced in
glia throughout brain by subarachnoid hemoglobin.
J Cereb Blood Flow Metab. In press.
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Abstract
Introduction
