A review of hemoglobin and the pathogenesis of cerebral vasospasm

RL Macdonald and BK Weir

Stroke. 1991;22:971-982
doi: 10.1161/01.STR.22.8.971
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 971

Progress Review

A Review of Hemoglobin and the

Pathogenesis of Cerebral Vasospasm
R. Loch Macdonald, MD, and Bryce K.A. Weir, MD

We believe that current experimental and clinical evidence can be most satisfactorily
interpreted by assuming that oxyhemoglobin is the cause of cerebral vasospasm that follows
subarachnoid hemorrhage. We review the pathogenetic mechanisms by which oxyhemoglobin
affects cerebral arteries. The relative importance of each of these mechanisms in the genesis of
vasospasm, the biochemical pathways of oxyhemoglobin-induced smooth muscle contraction,
and the intracellular actions of oxyhemoglobin on smooth muscle and on other cells in arteries
are still not definitely established. (Stroke 1991^2:971-982)

into the supernatant fluid starting after 2 days.917-23

C erebral vasospasm is an important cause of

cerebral ischemia and death1"3 following
aneurysmal subarachnoid hemorrhage
(SAH). Cerebral vasospasm is usually the most fre-
quent serious complication in survivors of SAH,1-3
although recent reports show that with hypervolemic
hypertensive therapy and calcium channel antago-
nists, morbidity and mortality from vasospasm may
have become relatively less important.4
Advances have also been made in identifying the
spasmogen responsible for vasospasm and in defining
how it causes arterial narrowing. Oxyhemoglobin
(OxyHb) is probably the principal pathogenetic
agent.5"7 We review evidence supporting the theory
that OxyHb causes vasospasm. This substance has
many mechanisms of action that may be important in
vasospasm. These include the release of free radi-
cals8-9; the initiation and propagation of lipid perox-
idation9; metabolism to bilirubin, which is another
potential spasmogen10-11; the release of vasoactive
eicosanoids12 and endothelin13 from the vessel wall;
perivascular nerve damage14-15; the inhibition of en-
dothelium-dependent relaxation16; and the induction
of structural damage in the arterial wall.15
Attempts to Isolate the Spasmogen
Incubation of blood in vitro results in slow hemo-
lysis of erythrocytes (RBCs) with release of OxyHb
From the Division of Neurosurgery, University of Alberta,
Edmonton, Canada.
Dr. Macdonald is a fellow of the Alberta Heritage Foundation
for Medical Research. This work was supported in part by grants
to Dr. Weir from the National Institutes of Health (5 R01
NS25946-03) and the Medical Research Council of Canada (MA
9158).
Address for correspondence: B.K.A. Weir, Professor and Chair-
man, Department of Surgery, University of Alberta, 2D1.02 W.C.
Mackenzie Health Sciences Center, Edmonton, Alberta, T6G 2B7,
Canada.
Received February 1, 1991; accepted April 3, 1991.
With time OxyHb is oxidized to methemoglo-
bin,91718-20 but further breakdown of heme to biliru-
bin occurs only in vivo.617-24 The exact mechanisms by
which heme groups of hemoglobin (Hb) are broken
down into bilirubin are not known, but they probably
require enzymes present only in living cells.25 After
SAH, RBCs remain fixed in the subarachnoid space
for days and they disappear by hemoryzing, similar to
the process in vitro.17-26
In 1944, Zucker27 recognized that RBC cytosol was
vasoactive although platelets and serum were much
more potent vasoconstrictors. Further experiments
have documented that RBCs contain a vasoactive
substance that is released by hemolysis.18"22-28"37
Humans with clinically significant vasospasm on
average have higher temperatures and more often
have leukocytosis than patients with ruptured aneu-
rysms without vasospasm.38 In experimental animals,
hemogenic meningitis, which may produce these
changes, is due to the heme component of RBCs.39'40
Many investigators have assayed the vasoactivity of
incubated and aged mixtures of whole blood, blood
components, and cerebrospinal fluid (CSF) on dog
and cat basilar arteries in vitro. 18-20,2231-33,41 Results
are remarkably concordant between experiments and
may be summarized as follows. Fresh serum, platelet-
rich plasma, and lysed RBCs have significant vasoac-
tivity whereas fresh, intact RBCs are inert. With
incubation, serum and platelet-rich plasma become
inactive whereas the contractile activity of intact or
h/sed RBCs persists. Similar experiments were per-
formed in vivo by Mcllhany and colleagues,35 who
injected vasoactive fractions of hemoryzed RBCs, as
collected on Sephadex G-75 gel filtration chromatog-
raphy, into the cisterna magna of dogs. Vasoconstric-
tion of the basilar artery was observed for 2 days.
Peterson et al42 found that RBC lysis was necessary

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972 Stroke Vol 22, No 8 August 1991
for development of vasospasm after the subarachnoid
injection of blood into dogs. In summary, during the
time of vasospasm in man, the vasoactivity of blood
incubated in vitro, when tested on the basilar artery
in vitro, resides in RBCs.
The role of RBCs in generating vasospasm has
been evaluated further by long-term in vivo studies.
The RBCs are the blood component necessary for
development of vasospasm in cats43 and dogs.44 May-
berg et al45 developed a piglet model whereby blood
components could be selectively applied to the mid-
dle cerebral artery (MCA) for 10 days. Whole blood
caused a significant reduction in lumen diameter.
Selective application of washed RBCs, RBC cytosol,
and pure porcine Hb also caused significant vaso-
spasm whereas leukocytes and platelet-rich plasma
did not.
The vasoactive substance released by RBCs has
been isolated by many groups.19-20-22^*1-33-36 The com-
pound has consistently been found to be a peptide
with a molecular weight, spectrophotometric absorp-
tion pattern, and movement on electrophoresis sim-
ilar or identical to those of OxyHb.19-20-22-31-33-36 Only
Cheung et al32 found a molecular weight (40,000-
45,000 d) that is low for Hb, which has a molecular
weight of 64,500 d.
These investigations show that RBCs are essential
for the development of vasospasm. Results of studies
isolating the spasmogen in RBCs and of long-term in
vivo studies43-45 point to OxyHb as the prime candi-
date to cause vasospasm.
Studies of Cerebrospinal Fluid After
Subarachnoid Hemorrhage
In man, vasospasm has its onset 3 days after SAH,
peaks after 6-7 days, and resolves by 14 days.46 This
unusual time course could be explained by the spas-
mogen responsible for vasospasm exhibiting a similar
time course of appearance in the CSF near arteries in
spasm. An alternative hypothesis explains the delay
in onset by suggesting that arterial narrowing is due
to another mechanism such asfibrosisand inflamma-
tory-cell infiltration of the arterial wall47 or to intimal
proliferation.48-49 In our opinion, this latter hypothe-
sis is untenable for a variety of reasons. There is no
evidence that arterial wall area increases enough
during vasospasm to narrow the lumen.4849 Intimal
proliferation and arterial wall fibrosis typically de-
velop after vasospasm resolves.49
Pathology of the subarachnoid space following hu-
man SAH shows that within 24 hours after SAH there
is intense porymorphonuclear cell infiltration of the
meninges. Phagocytosis and breakdown of RBCs oc-
cur by 16-32 hours. Breakdown of RBCs peaks
around day 7 but continues for days, with clumps of
intact RBCs still enmeshed in the arachnoid for up to
35 days after SAH. At 7 days, the inflammatory
reaction subsides and is composed equally of lympho-
cytes and phagocytes. After 10 days, fibrosis of the
subarachnoid space begins. Therefore, during the
time of vasospasm, the most prominent process occur-
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ring in the subarachnoid space is RBC hemolysis.50-52
Clearance of blood injected into the subarachnoid
space of animals is rapid, and most RBCs are removed
by methods other than hemolysis.40-53-55 This is prob-
ably why single injections of liquid blood in experi-
mental animals fail to mimic human vasospasm.
Hemolytic breakdown of RBCs observed micro-
scopically may also be observed by spectrophotomet-
ric examination of pigments released into the CSF.
Barrows and colleagues17 used absorption spec-
trophotometry to examine CSF after SAH; OxyHb
appeared 2 hours after SAH. By 4 days postictus
bilirubin was present, and over the following week
the amount of OxyHb decreased and that of bilirubin
increased. In the absence of further bleeding, both
pigments disappeared after 2-3 weeks. Bilirubin is
present in normal serum and could be observed in
the CSF immediately after massive SAH. In cases of
SAH methemoglobin was not detected, although it is
now believed to be present.56-57 Results of these and
other studies58"60 show that OxyHb is present during
vasospasm whereas small molecules and proteins
such as albumin, noradrenaline, and serotonin are
cleared rapidly from the CSF.53-55
Buckell61 first investigated the contractile activity
of CSF from patients with ruptured aneurysms. Some
CSF activity could be accounted for by serotonin, but
in half the cases other substances were present that
caused contraction. The contractile activity of xan-
thochromic CSF has been frequently studied.62-73
Allen et al62-63 suggested that the vasoactivity of
xanthochromic CSF was due to serotonin. Other
investigators,64-67-70-74 however, report that drugs that
block serotonin, histamine, acetylcholine, norepi-
nephrine, epinephrine, propranolol, and angiotensin
II do not prevent hemorrhagic CSF from contracting
cerebral arteries in vitro. Calcium channel antago-
nists have consistently at least partially blocked such
contractions.68-70 Evidence reviewed below indicates
that OxyHb-induced contractions show pharmaco-
logical properties similar to those of the agent in
xanthochromic CSF. Sasaki et al74 further delineated
the nature of the contractile activity by showing that
disulfide bond-reducing agents blocked contractions
induced by xanthochromic CSF in dog basilar artery.
Disulfide bond reducers are known to block smooth
muscle contraction due to prostaglandins (PGs), Hb,
and lipid peroxides. Yamamoto and colleagues75 iso-
lated a spasmogen from xanthochromic CSF that was
believed to possibly be endothelin, although the sys-
tem used to assess vasoactivity was unconventional.
If the vasoactivity in xanthochromic CSF is due to
OxyHb, then CSF OxyHb concentration should cor-
relate with severity of vasospasm. This relation has
been difficult to confirm, however, partly because
lumbar CSF OxyHb concentrations do not accurately
reflect concentrations adjacent to spastic arteries.76-77
Ohta and coworkers76 found that vasospasm did
correlate with the concentration of OxyHb in periar-
terial hematomas after SAH. An additional problem
with assays in vitro is that OxyHb may not be able to

exert all of its damaging effects in vitro. For example,
OxyHb would not be converted to bilirubin in vi-
tro,6'25 and its effects on the innervation of arteries
would probably be altered in vitro.78
Hemoglobin as a Vasoconstrictor
Vasospasm probably begins as smooth muscle con-
traction, but due to the prolonged and severe nature of
the contraction, vasoconstriction becomes temporarily
irreversible.48-4979 There may be changes in vessel wall
collagen and in the contractile apparatus of smooth
muscle that contribute to narrowing and cause stiffness
of the arterial wall.47'48-80-82 Morphological changes in
vasospasm and their relation to OxyHb are reviewed
below. Studies of the effects of Hb solutions on cerebral
arteries both in vitro and in vivo are therefore relevant
to theories implicating Hb as a cause of vasospasm.
Studies In Vitro
In vitro, Hb contracts isolated smooth muscle cells
and cerebral arteries of several different animal
species.9'12-21-34'76'79'83-104 Fujii and Fujitsu83 reported
that OxyHb contracted smooth muscle cells cultured
from rat aorta. Ultrastructural changes including
vacuolation, cell degeneration, and loss of internal
cell structure were noted after 24 hours of exposure.
We have used electrophysiology to study smooth
muscle cells isolated from rat cerebral arteries.84 A
solution containing OxyHb and small amounts of
methemoglobin contracted cells and increased calci-
um-dependent potassium currents. Cells died rapidly
after exposure. Pure methemoglobin solution did not
cause any of these effects.
Cook et al85 demonstrated that Hb causes slowly
developing and long-lasting contraction of dog cere-
bral arteries, rabbit ear arteries, and rat stomach
fundus in vitro. These workers used a solution of Hb
containing 20% OxyHb. Asano and associates9-21
performed similar experiments on dog basilar artery
in vitro and found that both OxyHb and methemo-
globin caused dose-dependent contractions, although
OxyHb was much more potent. These results are
consistent with many others.86-8791-103
Systemic and cerebral arteries have differing phar-
macological properties79; these arteries also vary in
their sensitivity to OxyHb, providing one reason for the
predilection of vasospasm for cerebral arteries.86-87'102
Studies In Vivo
Many studies have investigated the short-term
effects of Hb applied to cerebral arteries exposed in
105
situ.2o^6,76,io5-ii3 Tn i975 ; Chokyu exposed rat basi-
lar artery in situ and demonstrated a 30% constric-
tion due to the topical application of hemoh/sate of
RBCs and a 19% constriction due to pure OxyHb.
Similar to results in vitro, others have shown a similar
activity of OxyHb and have generally shown that
methemoglobin is much less vasoactive.20-107'112 Only
Fox106 reported that Hb did not contract canine
basilar artery exposed in situ, but it is unclear if the
Hb solution used contained OxyHb.
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Macdonald and Weir Hemoglobin and Vasospasm 973
Most of the above experiments confirm that OxyHb
causes vasoconstriction. Pure methemoglobin is proba-
bly inert, but the presence of small amounts of OxyHb
will render a solution vasoactive. Wellum et al78-88 have
expressed concern that OxyHb is not a strong enough
vasoconstrictor to cause severe vasospasm. However, if
OxyHb is responsible for vasospasm then short-term
experiments in vitro would not replicate conditions in
vivo, where arteries are exposed to high concentrations
of OxyHb for many days. The concentration of OxyHb
used in most in vitro and short-term in vivo studies
ranges from 10"8 to 10"2 M, with maximal contraction
developing at concentrations equal to and above 10~3
M, depending on the preparation.9-85-89-91-94'98-101 Mea-
surements of Hb concentrations in subarachnoid he-
matomas76 suggest that adequate amounts of OxyHb
are present near vasospastic arteries for long enough to
produce maximum contraction.
The issue of duration of exposure to the spas-
mogen has been addressed in experimental models of
vasospasm where changes in arterial diameter can be
assessed over many days. Single intracisternal injec-
tions of low doses of OxyHb have been given to small
numbers of dogs19-114-116 and baboons117 with vari-
able results but, unfortunately, single injections do
not produce a model that is similar to the human
condition, where large amounts of OxyHb are pres-
ent adjacent to cerebral arteries for days.76
Mayberg and colleagues45 developed a piglet
model of SAH that kept high concentrations of Hb or
various blood components adjacent to the right MCA
for 10 days. Pure porcine Hb (roughly 15% as
OxyHb) resulted in a significant decrease in lumen
area that was equal to that caused by RBC cytosol
containing a similar amount of Hb. Although whole
blood caused a greater decrease in lumen area than
did Hb, these authors suggested that this was because
the former was associated with more residual clot-
containing Hb at day 10. Therefore, all vasoactivity of
the whole blood was accounted for by Hb.
In our laboratory, cynomolgus moiikeys had cath-
eters placed along the right MCA and connected to
subcutaneous CSF reservoirs.6 In a randomized and
controlled study of 40 monkeys, multiple intrathecal
injections of OxyHb, methemoglobin, bilirubin, mock
CSF, or supernatant fluid from an incubated mixture
of autologous blood and mock CSF were given for 6
days. Significant vasospasm of the right MCA, as
judged by comparison of angiograms taken at base-
line and on day 7, developed in animals injected with
OxyHb and supernatant fluid. Pure methemoglobin
produced no significant arterial narrowing.
Mechanisms of
Oxyhemoglobin-Induced Vasoconstriction
Studies of How Oxyhemogbbin
Causes Vasoconstriction
Contraction caused by receptor-operated systems
usually decreases with time due to tachyphylaxis,
desensitization, and/or autoregulation.78-79 The time
974 Stroke Vol 22, No 8 August 1991
course of vasospasm suggests that smooth muscle
contraction by a conventional receptor-operated sys-
tem is not the sole mechanism responsible for arterial
narrowing. Most agents suggested to be mediators of
vasospasm, such as serotonin, biogenic amines, pep-
tides, and eicosanoids, however, probably act on cell
surface receptors.45-79118 Further, antagonists of re-
ceptor-operated vasoconstrictors, including atropine,
methysergide, cinanserin, ketanserin, phenoxybenz-
amine, phentolamine, mepyramine, chlorphe-
niramine, propranolol, saJbutamol, angiotensin, sar-
cosine, alanine, theophylline, and quinine, have
consistently failed to reverse OxyHb-induced con-
tractions of cerebral arteries in vitro and in
VJVO 3136,7635-87,94,93,98,103,107-109,112,119-121
Drugs reported to relax smooth muscle contracted
with OxyHb include papaverine, calcium channel
antagonists, and some inhibitors of eicosanoid syn-
thesis.85-86-95-108-109-119"121 The latter are discussed in
the next section. Although calcium channel antago-
nists partly reverse OxyHb-induced contraction of
cerebral vessels in vitro, these drugs do not dilate
monkey122 or human123124 cerebral arteries that are
vasospastic. Kajikawa et al108-109 found that after
exposing rat basilar arteries in situ to barium chloride
for 3 hours, papaverine did not dilate them, providing
evidence that the duration of contraction bears upon
the ability of pharmacological antagonists to relax
smooth muscle.
OxyHb acutely increases the intracellular concen-
tration of inositol phosphates, which are second
messengers involved in smooth muscle contraction.125
There are few other reports investigating the intra-
cellular mechanism of action of OxyHb, which is
obviously an important question.
Eicosanoids
Eicosanoids are products of enzymatic metabolism
of arachidonic acid and include PGs, thromboxanes,
and leukotrienes.126
After SAH in monkeys and dogs, vasospastic ves-
sels synthesize less PGI2 and more vasoconstricting
PGE2.126-130 Decreased vasodilator influence from
PGI2, combined with endothelial damage with adher-
ence of platelets and release of vasoconstricting PGs
and thromboxane A2, could contribute to vaso-
spasm.3-12-130 The effects of OxyHb on vessel wall
eicosanoid synthesis in vivo have been investigated by
Tokoro.129 He reported that injection of OxyHb into
dog cisterna magna decreased PGI2 levels in the
basilar artery 4 and 7 days later. There was no change
in the thromboxane A2 level in the arterial wall.
Studies show that following SAH in humans, the
CSF contains elevated levels of PGF^ and possibly
other PGs.126'131-133 Dog basilar artery removed 6 days
after SAH synthesizes more PGE^ than normal.127128
Experiments in vitro using bioassay systems to mea-
sure eicosanoids show that OxyHb releases vasoactive
PGs from several types of blood vessels,12-134-135 poten-
tially accounting for the changes seen after SAH.
Toda135 concluded that OxyHb-induced contraction
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was dependent on a mechanism related to the endo-
thelium but not involving endothelium-derived relax-
ing factor (EDRF). He suggested that PGs released
from endothelium by OxyHb were important in these
contractions.
The effects of inhibitors of PG and thromboxane
synthesis on experimental and human vasospasm
have been equivocal.92-135-139 Studies of OxyHb-in-
duced dog basilar artery contraction in vitro show
that indomethacin and aspirin, inhibitors of cyclooxy-
genase, have no effect on86-90-103 or only partially
prevent12-85-87-94-119 such contractions. Thromboxane
synthetase inhibitors were also ineffective in prevent-
ing OxyHb from contracting dog102 and guinea pig140
basilar artery.
Thus, although OxyHb affects vessel wall ei-
cosanoid production and could account for the alter-
ation in CSF levels of these substances after SAH,
inhibitors of synthesis of PGs and thromboxanes do
not prevent vasospasm. If OxyHb acts at multiple
sites in the vessel wall (e.g., direct action on smooth
muscle cells by the release of free radicals and the
production of lipid peroxides, combined with the
release of eicosanoids, EDRF, and endothelin from
the endothelium), then antagonizing only one of
these systems might not completely reverse the con-
traction. Furthermore, other factors, such as leuko-
trienes, which are also potent vasoactive agents and
the concentrations of which increase after SAH, have
not been thoroughly investigated.141-143
Free Radicals
OxyHb spontaneously autoxidizes to methemoglo-
bin, releasing superoxide anion radical (O2").8-144
Spectrophotometric studies show that after SAH,
hemolyzing subarachnoid RBCs release OxyHb,
which autoxidizes to methemoglobin, potentially re-
leasing O2" into the subarachnoid space.55-59 In
conjunction with the iron in Hb, O2" has been
postulated to initiate and propagate lipid peroxida-
tion by the Haber-Weiss reaction and Fenton chem-
istry.9-21 Lipid peroxides are known to cause vasocon-
striction and structural damage to cerebral arteries
both in vitro145-146 and in vivo.147'148
There is additional evidence for a role of lipid
peroxidation, and therefore OxyHb, in the genesis of
vasospasm. Vasoactivity of blood incubated in vitro
correlates with its concentration of OxyHb and with
its content of lipid peroxides, represented by thiobar-
bituric acid-reactive substances.9-21-147 Concentra-
tions of lipid peroxides in the CSF of patients with
SAH correlate with vasospasm.9-21-147-149-150 An inhib-
itor of iron-dependent lipid peroxidation, U74006F
significantly diminished vasospasm in a primate
model of SAH.151 Despite questions about the exact
mechanism of initiation of lipid peroxidation,152 de-
tection of 5-hydroxyeicosatetraenoic acid in CSF
after SAH shows that lipid peroxidation occurs.153
Although OxyHb probably does propagate lipid
peroxidation after SAH, it has not been shown in vivo
that the process is essential for the development of

vasospasm. This would require evidence that inhibi-
tors of lipid peroxidation prevent vasospasm151 and
evidence that lipid peroxidation precedes the devel-
opment of vasospasm since the arterial wall damage
that accompanies vasospasm would produce lipid per-
oxides.152 Furthermore, since vasospasm correlates
best with large volumes of subarachnoid blood,5-154 the
concentrations of lipid peroxides in CSF would be
elevated in association with vasospasm whether or not
their production is related to vasospasm.
Effects of free radical-scavenging enzymes on the
vasoactrvity of OxyHb on cerebral arteries in vitro have
been reported.36-84'90-91.9'*.109 Kamiyama and cowork-
ers110 found that superoxide dismutase, catalase, and
l,4-diazabicyclo[2.2.2]octane were effective inhibitors
of OxyHb-induced contraction of cat basilar artery
exposed in situ. Fujita et al90 found superoxide dismu-
tase to be ineffective at antagonizing OxyHb-induced
contraction of dog basilar artery in vitro. The effects of
catalase alone have also been inconsistent.36-91-94
Electrophysiological studies of isolated rat cere-
bral artery smooth muscle cells have shown OxyHb to
activate calcium-dependent potassium currents and
cause cell death.84 Catalase protected cells from
OxyHb, whereas superoxide dismutase did not. Xan-
thine and xanthine oxidase did not cause electrophys-
iological changes in cells, whereas the generation of
hydroxyl radical in the bathing solution was damag-
ing. The findings implicate the hydroxyl free radical
rather than other oxygen free radicals although it
may be difficult to define the actual free radicals
involved based on such scavenger experiments.152
In summary, superoxide dismutase and catalase
alone are poor antagonists of OxyHb-induced cere-
bral artery contraction. There are several possible
reasons for this. Free radical mechanisms may not be
important or they may not be the sole pathway in the
genesis of contraction by OxyHb. Superoxide dismu-
tase and catalase may not be able to penetrate
arterial walls to get to the locations where damaging
free radical reactions occur.152 Activity of both super-
oxide dismutase and a hydrogen peroxide scavenger
may be necessary to prevent the genesis of oxygen-
derived free radicals since superoxide dismutase pro-
duces hydrogen peroxide, which can form hydroxyl
radical in the presence of iron or ferrous pro-
teins.155-156 Catalase or glutathione peroxidase, if
present, would catabolize hydrogen peroxide, pre-
venting this reaction. Hydrogen peroxide157 and iron
ions158 can also inhibit superoxide dismutase.
Endothelium-Dependent Relaxation
Furchgott and Zawadzki159 showed that the va-
sodilatory action of acetylcholine on rabbit aorta
was mediated by release of an intermediate sub-
stance from endothelial cells. This substance has
been termed EDRF. Other vasodilators relax vas-
cular smooth muscle through this endothelium-
dependent mechanism, which has been subject to
recent review.160
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Macdonald and Weir Hemoglobin and Vasospasm 975
After SAH in dogs, vasospastic basilar arteries
contracted with PGF^ and uridine 5'-triphosphate
exhibit impaired relaxation to arginine vasopressin
and thrombin.161-162 Endothelium-dependent relax-
ation is also inhibited after SAH in rabbits163164 and
monkeys.165
OxyHb and other ferrous hemoproteins but
not methemoglobin or ferric hemoproteins are
well-known inhibitors of endothelium-dependent
relaxation in a number of vascular preparations in
vitro.16.95-104.121.140.160.166-173 Whether or not endo-
thelium-dependent relaxation occurs correlates
with levels of cyclic guanosine monophosphate in
the arterial wall.166
A study in vivo of endothelium-dependent relax-
ation and Hb was reported by Byrne et al.113 In pigs,
intracistenial injection of OxyHb caused acute vaso-
constriction of intrathecal arteries. Intracarotid infu-
sion of acetylcholine caused vasodilation before the
intracisternal injection of OxyHb and vasoconstric-
tion afterwards.
Kanamaru et al120 found that xanthochromic CSF
from SAH patients could inhibit A23187-induced
endothelium-dependent relaxation in dog basilar ar-
tery in vitro. The levels of OxyHb in CSF correlated
with the degree of inhibition of endothelium-depen-
dent relaxation.
The ability of OxyHb to inhibit endothelium-de-
pendent relaxation suggests that OxyHb is responsi-
ble for this event after SAH. How OxyHb prevents
endothelium-dependent relaxation has been investi-
gated. Both SAH and OxyHb damage arterial endo-
thelium, possibly decreasing EDRF synthesis by en-
dothelial cells.1548-174 Kim et al,161 however, used a
bioassay system for EDRF to show that EDRF
secretion by vasospastic dog basilar artery was not
impaired. EDRF may be nitric oxide,160 and Hb binds
nitric oxide with an affinity 1,500 times higher than its
affinity for oxygen.175 Thus Hb, which presumably
does not enter cells, could prevent EDRF, if it is
nitric oxide, from entering smooth muscle cells and
producing its effects.
The effects of OxyHb on EDRF have been assessed
in vitro using more physiological systems that isolate
the intraluminal and extraluminal aspects of the artery.
Although OxyHb applied extraluminally to dog and
rabbit cerebral arteries has been found to have little
effect on endothelium-dependent relaxation,170171
other researchers reported the inhibition of endotheli-
um-derived responses after exposing extraluminal sur-
faces of cerebral arteries to OxyHb.104-172
The effects of Hb on endothelium are further
complicated by observations that acetylcholine
causes transient hyperpolarization and relaxation of
rat aorta and guinea pig basilar artery precontracted
by noradrenaline.170 Hb prevents relaxation but
doesn't alter hyperpolarization. It has been hypoth-
esized that acetylcholine releases EDRF and endo-
thelium-derived hyperpolarizing factor from endo-
thelial cells.
976 Stroke Vol 22, No 8 August 1991
In addition to EDRF, endothelium-derived hyper-
polarizing factor, and eicosanoids, endothelial cells
synthesize a potent vasoconstrictor peptide, endothe-
lin.176 Vasospasm is associated with increased CSF
levels of endothelin.177-179 Findings that OxyHb aug-
ments the release of endothelin from cultured bovine
cerebral artery endothelial cells suggest that OxyHb
may be responsible for elevating CSF endothelin
levels after SAH.13
The importance of the endothelium in vasospasm
remains unclear, although OxyHb contracts cerebral
arteries denuded of endothelium, suggesting that
endothelin release is not requisite for vaso-
spasm.96.99.100.103.119.180 Further, if the endothelium is
removed after arteries are contracted with OxyHb,
relaxation does not occur.104
Bilirubin
In 1949, Jackson39 injected bilirubin into the cis-
terna magna of dogs, producing severe inflammatory
reactions with fever, malaise, and increased CSF
protein and leukocyte concentrations. The reaction
was comparable to that induced by OxyHb, and he
believed that one of these two agents caused he-
mogenic meningitis. Angiography was not done.
Several early experiments found that bilirubin had
no effect on cerebral artery diameters in vitro.105107
In these studies, bilirubin was applied to arteries for
minutes only. Duff and colleagues10 reported that
bilirubin solutions caused progressive vasoconstric-
tion of cat and baboon basilar arteries. Electron
microscopy of the arteries showed swelling of endo-
thelial cells, degeneration of axons and varicosities in
the adventitia, and extensive vacuolation of smooth
muscle and endothelial cells. These results were
supported by investigations by Miao and Lee11 that
demonstrated the vasocontractile effects of bilirubin
on cerebral arteries.
Intrathecal injection of bilirubin for 6 days was
performed in our laboratory as part of a randomized
trial to study the role of various blood components in
the etiology of vasospasm in monkeys.6 Bilirubin
caused a nonsignificant 13% decrease in right MCA
diameter. Ultrastructural examination of these arter-
ies did show some pathological changes.
Although the time course of appearance of biliru-
bin in the CSF after SAH is similar to that of
vasospasm,17-56 there are several reasons to doubt
that bilirubin is an important spasmogen. Bilirubin
contaminates CSF in other diseases such as neonatal
and obstructive jaundice.58-181-183 Focal neurological
deficits, which might be related to vasospasm, have
rarely been noted in these conditions.184 Wahlgren
and Bergstrom58 found concentrations of bilirubin in
the CSF of patients with obstructive jaundice that
caused vasospasm in the study of Duff et al.10 Bili-
rubin, especially when bound to albumin, possesses
antioxidant activity and inhibits lipid peroxida-
tion.183-185186 Furthermore, in the subarachnoid
space bilirubin is produced by an enzyme system with
limited capacity, which may result in bilirubin con-
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centrations that are too low to induce significant
arterial narrowing after SAH.187 Findings that in-
trathecal injections of OxyHb cause vasospasm
whereas injections of methemoglobin do not also cast
doubt on the bilirubin theory since both types of Hb
produce bilirubin in the subarachnoid space.6-25
Neuwgenic Effects
Cerebral arteries receive adrenergic, cholinergic,
and peptidergic innervation and possess receptors for
neurotransmitters such as serotonin, a- and £-adren-
ergic drugs, dopamine, and histamine although the
precise role of these nerves and receptors in the
regulation of cerebrovascular tone is unknown.188-190
Adventitial nerve endings in cerebral arteries degen-
erate after experimental SAH,14-15-48-190-191 and this is
accompanied by a transient loss of catecholamine histo-
fluorescence around cerebral arteries.192-193 Disappear-
ance of perivascular nerves after SAH, however, did
not correlate with vasospasm in rats194 and primates.195
Sympathectomy can have no effect on196 or it can
alleviate197 vasospasm in experimental animals and
humans. Drugs that block a-adrenergic, ^-adrenergic,
and muscarinic receptors have little effect on cerebral
vasospasm induced by whole blood198 and on vasocon-
striction due to OxyHb.30-85-86-103.112-119.121
Despite contradictory evidence regarding the sig-
nificance of changes in cerebrovascular innervation
in relation to vasospasm, SAH clearly does damage
these nerves and OxyHb may be responsible for this
degeneration.15-173-199 Inhibition of vasodilator nerves
and potentiation of vasoconstricting nerves by OxyHb
may create a favorable setting for the development of
arterial narrowing after SAH.173 Few studies in vivo
have been reported and obviously, results might be
different when arteries are in situ with anatomically
intact innervation. The effects of OxyHb on peptider-
gic innervation of cerebral arteries have not been
investigated in detail.189
Synergistic and Other Effects
OxyHb accentuates the contractile effects of hypox-
ia,1999 serotonin,97-102 potassium,140 and fibrin degra-
dation products39 on cerebral arteries in vitro. Recent
investigations, however, have suggested that Hb, and
presumably OxyHb, is a sufficient cause of vaso-
spasm.6'45 White118 believed that PGs were probably
important in vasospasm and that serotonin, plasmin,
and antithrombin III were possibly involved. Other
major contenders for the cause of vasospasm include
bilirubin,10'11 endothelin,13177-179 and lipid perox-
ides.9-21 Since OxyHb is metabolized to bilirubin,25 is a
potent propagator of lipid peroxidation,9-21 and aug-
ments the release of eicosanoids and endothelin from
arteries,12-13'94 consideration of any one of these sub-
stances as the sole cause of vasospasm may be a moot
point. Clearly, OxyHb, while potentially the only agent
necessary to precipitate vasospasm, acts by diverse
mechanisms to cause arterial narrowing.

Hemoglobin and Pathological Changes in
Cerebral Arteries
Most investigators agree that vasospasm is, at least
initially, due to smooth muscle contraction.45'47"49-174
Vasospastic arteries, however, are abnormal pharma-
cologically47.80-82^00-201 and ultrastructurally,26-47-49-174
indicating that processes occur during vasospasm
that are not simple muscular contraction. Regardless
of the significance of these changes, if OxyHb causes
vasospasm, then it should produce the particular
arterial wall changes that have been observed in
association with vasospasm.
Okada et al15 injected OxyHb into feline prepontine
cistern, causing myonecrosis, transformation of nerve
endings, invasion of myointimal cells into the tunica
intima, changes in endothelial cell basement mem-
brane, and detachment of endothelial cells after 24
hours. Kajikawa and colleagues108-109 applied OxyHb
to exposed rat basilar arteries for 3 hours. Vasospasm
resulted, and at electron microscopy endothelial cell
craters, blebs, and vacuoles were seen. Smooth muscle
cells developed vacuoles, nuclear pyknosis, and mito-
chondrial degeneration. OxyHb caused vacuolation
and degeneration of cultured rat aortic smooth muscle
cells, whereas serotonin- and norepinephrine-exposed
cells were normal.83
A spectrum of pathological changes have also been
produced by the exposure of cerebral arteries in vivo
to OxyHb.6-45 Therefore, OxyHb can produce mor-
phological changes in cerebral arteries that are indis-
tinguishable from those due to SAH.
Summary
There is good evidence that the release of OxyHb
from lysing subarachnoid RBCs is a key mechanism
responsible for vasospasm. OxyHb is present in high
concentrations in CSF during the time of vasospasm.
RBCs are the component of blood necessary for
vasospasm to develop, and the most vasoactive sub-
stance within RBCs is OxyHb. Although it is not as
potent a constrictor as some other agents, OxyHb can
act by a variety of pathways over a prolonged period
to produce arterial narrowing and ultrastructural
damage to arteries that are akin to those occurring
after SAH. While OxyHb may be the sole initiator of
vasospasm, the pathogenesis of action of OxyHb is
probably multifactorial, involving direct effects on
smooth muscle, release of vasoactive eicosanoids and
endothelin from the arterial wall, inhibition of endo-
thelium-dependent relaxation, production of biliru-
bin and lipid peroxides, and damage to perivascular
nerves.
The relative contributions of known mechanisms
of action of OxyHb to the pathogenesis of vasospasm
are not worked out. How OxyHb causes smooth
muscle contraction and the biochemical derange-
ments that it produces in smooth muscle are also key
unanswered questions.
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Macdonald and Weir Hemoglobin and Vasospasm 977
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KEY WORDS • cerebral arteries • cerebral vasospasm •
hemoglobin • subarachnoid hemorrhage