J Neurosurg 96:287–293, 2002

Brain edema after experimental intracerebral hemorrhage:

role of hemoglobin degradation products

FENG-PING HUANG, M.D., GUOHUA XI, M.D., RICHARD F. KEEP, PH.D., YA HUA, M.D.,
ANDREI NEMOIANU, B.A., AND JULIAN T. HOFF, M.D.
Departments of Neurosurgery and Physiology, University of Michigan, Ann Arbor, Michigan

Object. The mechanisms involved in brain edema formation following intracerebral hemorrhage (ICH) have not
been fully elucidated. The authors have found that red blood cell lysis plays an important role in edema develop-
ment after ICH. In the present study, they sought to determine whether degradation products of hemoglobin cause
brain edema.
Methods. Hemoglobin, hemin, bilirubin, or FeCl2 were infused with stereotactic guidance into the right basal gan-
glia of Sprague–Dawley rats. The animals were killed 24 hours later to determine brain water and ion contents.
Western blot analysis and immunohistochemistry were applied for heme oxygenase-1 (HO-1) measurement. The ef-
fects of an HO inhibitor, tin-protoporphyrin (SnPP), and the iron chelator deferoxamine, on hemoglobin-induced
brain edema were also examined.
Intracerebral infusion of hemoglobin, hemin, bilirubin, or FeCl2 caused an increase in brain water content at 24
hours. The HO-1 was upregulated after hemoglobin infusion and HO inhibition by SnPP-attenuated hemoglobin-in-
duced edema. Brain edema induced by hemoglobin was also attenuated by the intraperitoneal injection of 500 mg/kg
deferoxamine.
Conclusions. Hemoglobin causes brain edema, at least in part, through its degradation products. Limiting hemo-
globin degradation coupled with the use of iron chelators may be a novel therapeutic approach to limit brain edema
after ICH.

KEY WORDS • hemoglobin • iron • bilirubin • cerebral hemorrhage •

brain edema • rat

the United States, 15% of deaths due to cerebrovas-
I
N
cular disease result from spontaneous ICH. Many pa-
tients with an intracerebral hematoma deteriorate pro-
gressively because of secondary brain edema formation.15
A better understanding of the pathophysiology of edema
development after ICH may lead to better clinical manage-
ment of these patients.
Recently, new details of the pathophysiological mecha-
nisms involved in edema formation after ICH have been
reported. These include clot retraction in the first several
hours, the coagulation cascade and thrombin formation in
the 1st day, and RBC lysis and hemoglobin neurotoxicity a
few days later.4,22–25,48,51,54 Because hemoglobin-induced ede-
ma formation is triggered a few days after ICH, therapy tar-
geted at hemoglobin and its degradation products may offer
a new approach to therapy for ICH.
Hemoglobin breakdown products may have a key role in
the formation of brain edema. Metabolism of hemoglobin
by HO after RBC lysis results in the release of iron, carbon
monoxide, and biliverdin.27 Subsequently, biliverdin reduc-
tase catalyzes the conversion of biliverdin to bilirubin.20
Abbreviations used in this paper: HO = heme oxygenase; ICH =
intracerebral hemorrhage; NIH = National Institutes of Health;
RBC = red blood cell; SD = standard deviation; SnPP = tin-proto-
porphyrin.
Both enzymes are found in the brain,8 and inhibition of HO
is associated with attenuation of brain edema after ICH.47
Intracortical injection of iron, a potent catalyst for lip-
id peroxidation, causes focal epileptiform discharges and
brain edema.49,50 Hemoglobin induces neuronal death in
vitro, a process that is blocked by deferoxamine, an iron
chelator.36 In addition, bilirubin is toxic to brain tissue, al-
though it is considered to be an antioxidant at low concen-
trations.29,42
In this study, we examined the effects of hemoglobin
degradation products on the formation of brain edema. We
also studied the effects of an HO inhibitor, SnPP, and the
iron chelator deferoxamine, on hemoglobin-induced brain
edema. Upregulation of HO-1 protein was examined using
Western blot analysis and immunohistochemistry.
Materials and Methods
Animal Preparation and Intracerebral Infusion
The protocols for these animal studies were approved by the Uni-
versity of Michigan Committee on the Use and Care of Animals. A
total of 72 male Sprague–Dawley rats weighing 300 to 400 g were
used in this study. The animals were anesthetized with 40 mg/kg
intraperitoneally administered pentobarbital. The right femoral ar-
tery was catheterized for continuous blood pressure monitoring and

J. Neurosurg. / Volume 96 / February, 2002 287


TABLE 1
Physiological parameters in rats during intracerebral infusions*
Variable Value
MABP (mm Hg) 102  8
pH 7.43  0.03
PaO2 (mm Hg) 91  5
PaCO2 (mm Hg) 41  4
hematocrit (%) 41  1
glucose (mM) 7.3  0.2
* Values are expressed as the means  SD. Abbreviation: MABP = mean
arterial blood pressure.
blood sampling. Blood was obtained from the catheter for analysis
of pH, PaO2, PaCO2, hematocrit, and blood glucose. The rats’ core
temperature was maintained at 37.5˚C with the use of a feedback-
controlled heating pad. The rats were positioned in a stereotactic
head frame, and a 1-mm burr hole was made on the right coronal
suture 4 mm lateral to the bregma. A variety of solutions (30 l)
were infused with a microinfusion pump into the right caudate nu-
cleus through a 26-gauge needle (coordinates: 0.2 mm anterior, 5.5
mm ventral, and 4 mm lateral to the bregma) at a rate of 2 l/minute.
After infusion, the needle was removed and the skin closed with su-
tures.
Experimental Groups
The study was performed in four parts. In the first part we eval-
uated the effect of an intracerebral infusion of hemoglobin and its
degradation products (hemin, bilirubin, and iron) on brain edema
formation. In the second part we focused specifically on the involve-
ment of HO in brain edema formation after an intracerebral infusion
of hemoglobin. The effect of the HO inhibitor SnPP on hemoglobin-
induced brain edema was investigated in this part. In the third part
we studied the effects of the iron chelator deferoxamine, injected in-
traperitoneally, on hemoglobin-induced brain edema. In the fourth
part we examined HO-1 expression in the brain after hemoglobin in-
fusion.
Part 1. Five groups of five animals were used in the first part of
the study. Each rat received a 30-l infusion of normal saline, bo-
vine hemoglobin (300 mg/ml in saline), hemin (12 mg/ml in saline),
bilirubin (12 mg/ml in saline), or FeCl2 (10 mM in saline). The con-
centrations of injected hemoglobin breakdown products were the
same as the concentrations found in rat RBCs. The animals were an-
esthetized again and decapitated 24 hours after infusion. Brain wa-
ter, sodium, and potassium contents were measured.
Part 2. Four groups of five rats each were used in this part.
These four groups received an infusion of 30 l of saline, vehicle
(NaOH  HCl  saline, pH 7.4), hemoglobin (300 mg/ml), or he-
moglobin  SnPP (90 nM). The rats were killed 24 hours later to ex-
amine brain water and ion contents.
Part 3. Three groups of five rats each were studied in this part. All
the rats received a 30-l infusion of hemoglobin, followed immedi-
ately by intraperitoneal infusion of 1 ml saline, 50 mg/kg deferox-
amine, or 500 mg/kg deferoxamine (both dissolved in 1 ml saline).
The animals were decapitated 24 hours later to determine brain wa-
ter and ion contents.
Part 4. Two groups of six rats each either underwent a sham oper-
ation or received a 30-l hemoglobin infusion. The rats were killed
24 hours later for Western blot analysis (three rats per group) or im-
munohistochemical studies (three rats per group).
Brain Water and Sodium Contents
Animals were anesthetized and decapitated as described in our
previous studies.51,54 The brains were removed, and a 3-mm thick
coronal brain slice was cut 4 mm from the frontal pole. This section
of brain was divided into its two hemispheres along the midline, and
the cortex was separated from the basal ganglia bilaterally. The cere-
288
F. P. Huang, et al.
FIG. 1. Photograph showing a coronal section of rat brain ob-
tained 24 hours after a 30-l hemoglobin infusion.
bellum was also detached to serve as control tissue. The brain sam-
ples were immediately weighed on an electronic analytical balance
to obtain the wet weight. The samples were then dried in a gravity
oven at 100˚C for 24 hours to obtain the dry weight. Water contents
were expressed as a percentage of wet weight; the formula for calcu-
lation was (WW  DW)/WW. The dehydrated samples were digest-
ed in 1 ml of 1 M nitric acid for 1 week, and the sodium content of
this solution was measured with an automatic flame photometer. Ion
content was expressed in milliequivalents per kilogram of dehydrat-
ed brain tissue (mEq/kg dry wt).
Use of SnPP
The SnPP was dissolved in normal saline with 1 M NaOH to ob-
tain solutions at pH 8, and subsequently buffered with 6 M HCl to
pH 7.4 for final injection solutions. The solutions (100 mM) were
protected from light and freshly prepared just before use.
Western Blot Analysis
Animals were reanesthetized and underwent transcardiac perfu-
sion with saline. Brain tissues were sampled as described in Brain
Water and Sodium Contents. The rats were decapitated and a coro-
nal brain slice was cut as described for measurements of water con-
tent. The brain tissues were immersed in 0.5 ml Western blot sam-
ple buffer and were sonicated for 10 seconds. Twenty microliters of
the sample solution was taken for the protein assay. Western blot
analysis was performed as described previously.52 Briefly, 50 g of
protein was run on 10% polyacrylamide gels with a 4% stacking gel.
The protein was transferred to a hybond-C pure nitrocellulose mem-
brane. The membranes were probed with a 1:1000 dilution of the
polyclonal rabbit anti–rat HO-1 primary antibody. The membranes
were immunoprobed again with a 1:2000 dilution of the second an-
tibody (peroxidase-conjugated goat anti–rabbit immunoglobulin
G). Finally, the antigen-antibody complexes were visualized with a
chemiluminescence system and exposed to Kodak film. The relative
densities of HO-1 protein bands were analyzed with a public-do-
main imaging software program.
Immunohistochemical Studies
The rats were reanesthetized with 60 mg/kg intraperitoneally ad-
ministered pentobarbital and perfused with 4% paraformaldehyde in
0.1 M phosphate-buffered saline (pH 7.4). The brains were removed
and kept in 4% paraformaldehyde for 6 hours, then immersed in
25% sucrose for 3 to 4 days at 4˚C. The brains were embedded in
optimal cutting temperature compound and 18-m-thick sections
were made with a cryostat. The sections were incubated according
to the avidin–biotin complex method.52 The primary antibody was
rabbit anti–rat HO-1 (1:800 dilution). Normal rabbit immunoglobu-
lin G was used as a negative control.
J. Neurosurg. / Volume 96 / February, 2002
Brain edema after experimental intracerebral hemorrhage

Statistical Analysis
All data in this study are presented as the means
SD. Data from
different animal groups were analyzed using analysis of variance
with a Scheffé F-test or Student t-test. The differences were consid-
ered significant at probability values less than 0.05.
Sources of Supplies and Equipment
The Sprague–Dawley rats were purchased from Charles River
Laboratories, Portage, MI. The stereotactic head frame was man-
ufactured by Kopf Instruments, Tujunga, CA. The microinfusion
pump was acquired from Harvard Apparatus, Inc., S. Natick, MA.
The bovine hemoglobin was obtained from ICN Biomedicals, Inc.,
Aurora, OH. The hemin, bilirubin, and FeCl2 were supplied by Sig-
ma Chemical Co., St. Louis, MO. The SnPP was purchased from
Porphyrin Products, Inc., Logan, UT.
The electronic analytical balance (model AE 100) was acquired
from Mettler Instrument Co., Hightstown, NJ. The automatic flame
photometer (model IL943) was purchased from Instrumentation Lab-
oratory, Inc., Lexington, MA. The protein assay kit was obtained
from BioRad, Hercules, CA. The hybond-C nitrocellulose membrane
and the ECL chemiluminescence system were supplied by Amer-
sham International, Buckinghamshire, UK. The Kodak X-OMAT
film was manufactured by Eastman Kodak, Rochester, NY. The NIH
Image software (version 1.61) was obtained from the NIH, Bethes-
da, MD. The primary antibody (rabbit anti–rat HO-1) was acquired
from StressGen, Victoria, BC, Canada. The optimal cutting temper-
ature compound was obtained from Sakura Finetek U.S.A. Inc., Tor-
rance, CA.

Results
Physiological parameters in all animal groups were
recorded during intracerebral infusions. All physiological
variables, including mean arterial blood pressure, blood
pH, blood gases, hematocrit, and blood glucose, were con-
trolled within normal ranges (Table 1).
At 24 hours after the intracerebral infusion of 30 l of
hemoglobin, brain swelling was found in the ipsilateral
hemisphere, along with a midline shift (Fig. 1). Gross in-
spection of the brain revealed that most rats (four of five)
in the hemoglobin infusion group developed transtentorial
herniation. Herniation was not found in any animals in the
control group, however.
Brain water content was increased in the ipsilateral basal
ganglia 24 hours after intracerebral infusions of hemoglo-
bin, hemin, bilirubin, and FeCl2 (82.2
1.3%, 82.8
2%,
81.8
2.4%, and 83.5
2.1%, respectively, compared
with 77.9
0.2% in the saline control; p  0.01; Fig. 2A).
Edema formation after hemoglobin, hemin, bilirubin, or
FeCl2 infusions was associated with an accumulation of so-
dium ions (Fig. 2B).
Intracerebral coinjection of SnPP (90 nM) with hemo-
globin attenuated hemoglobin-induced brain edema in
the ipsilateral basal ganglia (80.2
0.8% compared with
82.4
1% in the hemoglobin group, p  0.01) and cortex
(80.5
0.8% compared with 82.3
1.6% in the hemo-
globin group, p  0.05) at 24 hours. We found that SnPP
had no effect on brain water content in the contralateral
hemisphere and cerebellum (Fig. 3).
Hemoglobin-induced brain edema was reduced in the
ipsilateral basal ganglia (80.5
1% compared with 82.4

1.1%, p  0.01) and cortex (80
0.2% compared with
82.3
1.3% in the control) by a large dose (500 mg/kg)
of deferoxamine injected intraperitoneally. A low dose (50
mg/kg) of deferoxamine, however, had no significant ef-
FIG. 2. Bar graphs showing brain water (A) and sodium ion con-
tent (B) 24 hours after infusion of 30 l of hemoglobin, hemin,
bilirubin, or FeCl2. Values are expressed as the means
SD. #p 
0.01 compared with the saline group.
fect on brain edema. The contralateral and cerebellar water
contents were not affected by either deferoxamine dosage
(Fig. 4).
In the normal brain, HO-1 protein was undetectable by
Western blot analysis (data not shown). After an infusion of
hemoglobin, however, HO-1 protein was upregulated in the
ipsilateral basal ganglia at 24 hours (3575
850 pixels
compared with 829
369 pixels in saline control, p 
0.01; Fig. 5). Immunohistochemical studies also detected
numerous HO-1 positive cells in the ipsilateral hemisphere
24 hours after hemoglobin infusion, whereas few HO-1
positive cells were found in the control group and in the
contralateral hemisphere (Fig. 6).
Discussion
In this study we demonstrate that an intracerebral infu-
sion of hemoglobin and its degradation products hemin,
iron, and bilirubin, cause the formation of brain edema
within 24 hours. Hemoglobin itself induces HO-1 upreg-
ulation in the brain, and HO inhibition by SnPP reduces
hemoglobin-induced brain edema. In addition, an intraperi-

J. Neurosurg. / Volume 96 / February, 2002 289


FIG. 3. Bar graph showing brain water contents in rats 24 hours
after infusion of 30 l of saline, vehicle, hemoglobin (Hb), or
hemoglobin and SnPP. Values are expressed as the means
SD.
*p  0.01 compared with saline or vehicle; **p  0.05 compared
with hemoglobin and SnPP; #p  0.01 compared with the other
groups.
toneal injection of a large dose of the iron chelator defer-
oxamine attenuates brain edema induced by hemoglobin.
These results indicate that hemoglobin causes brain injury
both by itself and through its breakdown products.
In a previous study51 it was demonstrated that an intrace-
rebral infusion of lysed RBCs results in marked brain ede-
ma formation. This edema formation appears to be mediat-
ed by hemoglobin, because an intracerebral infusion of rat
hemoglobin at concentrations found in erythrocytes also
results in marked increases in brain water content. The os-
molality of the infusate apparently does not contribute to
the increases of brain water content, because no brain ede-
FIG. 4. Bar graph showing brain water content in rats 24 hours
after infusion of 30 l of hemoglobin (Hb). The animals received
intraperitoneal injections of either 1 ml saline (control) or 50 mg/
kg or 500 mg/kg deferoxamine ([DFX] in 1 ml saline) right after
intracerebral infusion of hemoglobin. Values are expressed as the
means
SD. #p  0.01 compared with the saline group.
290
F. P. Huang, et al.
FIG. 5. Upper: The HO-1 levels were semiquantitated using
Western blot analysis 24 hours after either saline control (lanes 1 to
3) or intracerebral infusion of hemoglobin (lanes 4 to 6). Lower:
Bar graph depicting the results of Western blot analysis. Values are
expressed as the means
SD. #p  0.01 compared with control.
ma was found 2 hours after hemoglobin infusion. Osmotic
equilibration occurs within approximately 30 minutes in
the rat (Xi, et al., unpublished data).
Other studies indicate that hemoglobin has other dele-
terious effects on the brain. For example, an intracortical
hemoglobin injection in rats produces chronic focal spike
activity and gliosis.37 Hemoglobin inhibits Na/K adeno-
sine triphosphate activity,38 activates lipid peroxidation,9
exacerbates excitotoxic injury in cortical cell culture,35
and induces depolarization in hippocampal CA1 neurons.56
Koenig and Meyerhoff18 found that hemoglobin, at a con-
centration of 25 nM, induces the death of 50% of forebrain
neurons in culture within 8 hours and 72% within 24 hours.
A recent study in humans indicates that delayed brain
edema after ICH is associated with a significant midline
shift.57 We believe that this delayed edema (in the 2nd or
3rd weeks after onset of ICH in humans) is probably due to
erythrocyte lysis and hemoglobin-induced brain damage.
Oxyhemoglobin is a spasmogen that has been implicat-
ed in cerebral vasospasm after subarachnoid hemorrhage.26
In terms of ICH, an intraparenchymal infusion of lysed
RBCs (oxyhemoglobin) failed to reduce cerebral blood
flow in rats.53 In addition, ICH did not cause marked reduc-
tions of cerebral blood flow in animals.33,34,55 Finally, an in-
tracerebral infusion of methemoglobin mimics the effects
of lysed erythrocytes on edema formation.51
In this study we have shown that intracerebral injection
of hemin induces edema formation, indicating that this part
of the hemoglobin molecule can cause brain injury. In addi-
tion, our study indicates that heme breakdown products that
appear as the clot lyses play a major role in the formation
of brain edema. Heme from hemoglobin is degraded by HO
in the brain into iron, carbon monoxide, and biliverdin;
the latter is then converted to bilirubin by biliverdin reduc-
J. Neurosurg. / Volume 96 / February, 2002
Brain edema after experimental intracerebral hemorrhage
FIG. 6. Photomicrographs of rat brain sections showing HO-1 immunoactivities in the ipsilateral basal ganglia after
sham operation (A) and hemoglobin infusion (B). Examples of HO-1 positive cells are indicated by arrows. Bar = 20 m.
tase.20 Data from in vitro studies have shown that brief ex-
posures (1–2 hours) of neurons to hemoglobin are not tox-
ic, but their exposure to hemoglobin for 24 hours or more
causes neuronal death.36 This delayed effect of hemoglobin
may indicate that hemoglobin-induced brain injury results
from its breakdown products.
Our study shows that an intracerebral infusion of iron
causes brain edema, and that deferoxamine reduces hemo-
globin-induced edema, indicating that iron plays an impor-
tant role in edema formation after ICH. A cortical injection
of iron causes focal epileptiform paroxysmal discharges.10,50
Iron and lipid peroxidation also have an important role in
hemoglobin-induced brain injury. For example, a subpial
injection of FeCl2 induces edema and lipid peroxidation in
the brain.49 Iron can also stimulate the formation of free
radicals, leading to neuronal damage; it is known that fer-
rous (Fe2) and ferric (Fe3) iron react with lipid hydro-
peroxides to produce free radicals.40 Deferoxamine, an iron
chelator, can cross the blood–brain barrier,16 and data from
in vitro studies have shown that this substance reduces he-
moglobin-induced brain Na/K adenosine triphosphate
inhibition and neuronal toxicity.36,38 Furthermore, antioxi-
dants block neuronal toxicity induced by hemoglobin and
iron.3,36
Bilirubin is toxic to the brain although it is considered to
be an antioxidant. Fifty years ago, Jackson13 injected biliru-
bin into the cisterna magna of dogs, causing severe inflam-
matory reactions. In more recent studies it has been demon-
strated that bilirubin exerts many pernicious effects that
cause brain injury, including inhibition of phosphorylation
of the synaptic vesicle-associated protein synapsin I,11 dis-
turbance in high-energy phosphate levels,12 reduction in
mitochondrial activity, and disturbances in DNA synthesis,
protein synthesis, and ion transport.2 Amit and Brenner1
found that bilirubin is toxic to neurons and astrocytes in
vitro. Our results also indicate that bilirubin contributes to
brain edema formation after ICH. It should be noted, how-
ever, that micromolar concentrations of bilirubin may also
act as an antioxidant.30,42 Interruption of HO activity, which
reduces bilirubin and free iron production, has provided
a protective effect against hemorrhagic,47 ischemic,14 and
traumatic brain injury.32
Heme oxygenase consists of three enzymes, HO-1, HO-
J. Neurosurg. / Volume 96 / February, 2002
2, and HO-3.27,28 These forms have the following character-
istics: HO-1, which has also been called heat shock protein
32, is induced by a variety of stimuli;7,17 HO-2, the consti-
tutive isoform of HO, is localized mainly in the brain and
testes;43,45 and HO-3 protein is a poor heme catalyst and is
also found in the brain.28 Recent studies indicate that HO-1
upregulation increases free redox active iron production,
which may cause cell injury,21,44 although other studies indi-
cate that HO-1 plays an important role in cytoprotection
against oxidative injury.19,31,39
Our results demonstrate that hemoglobin upregulates
HO-1 protein levels in the brain and that inhibition of HO
by SnPP reduces hemoglobin-induced brain edema. From
these results we infer that upregulation of HO-1 in a heme-
rich environment that contains a high level of HO-2, which
occurs in the perihematomal zone, might cause excess free
iron and bilirubin accumulation, resulting in brain injury.
This finding is supported by Suttner and Dennery,44 who
found that HO-1 overexpression and reactive iron accumu-
lation are associated with oxygen cytotoxicity. In addition,
Lamb, et al.,21 found that hemoglobin stimulates lipid per-
oxidation in microsomal and cellular systems and that per-
oxidation is reduced by HO inhibitors and iron chelators.
When the concentration of heme products is not signif-
icantly increased, such as in cerebral ischemia, however,
HO produces a low concentration of bilirubin and is neuro-
protective.6
Carbon monoxide is another product generated in the
course of heme degradation by HO activity. Carbon mon-
oxide is a free radical that causes brain injury that is analo-
gous to nitric oxide–mediated damage.5,41,46
Conclusions
After ICH, hemoglobin, the main product of lysed
RBCs, plays a major role in delayed brain edema formation
through its degradation products. Limiting hemoglobin
degradation by HO inhibition and using iron chelators may
be useful treatments for ICH.
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Manuscript received April 16, 2001.
Accepted in final form September 25, 2001.
This study was supported by Grant Nos. NS-17760 and NS-
39866 from the NIH to Drs. Hoff and Xi, respectively.
Address reprint requests to: Guohua Xi, M.D., Room 5550
Kresge I, University of Michigan, Ann Arbor, Michigan 48109–
0532. email: guohuaxi@umich.edu.
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