Hydrogen Sulfide Is a Mediator of Cerebral

Ischemic Damage
Kun Qu, BSc; Christopher P.L.H. Chen, BMBCh, FRCP; Barry Halliwell, DPhil, DSc;
Philip K. Moore, PhD; Peter T.-H. Wong, PhD

Background and PurposeWe observed recently that elevated plasma cysteine levels are associated with poor clinical
outcome in acute stroke patients. In a rat stroke model, cysteine administration increased the infarct volume apparently
via its conversion to hydrogen sulfide (H2S). We therefore investigated the effects of H2S and the inhibition of its
formation on stroke.
MethodsCerebral ischemia was studied in a rat stroke model created by permanent occlusion of the middle cerebral
artery (MCAO). The resultant infarct volume was measured 24 hours after occlusion.
ResultsAdministration of sodium hydrosulfide (NaHS, an H2S donor) significantly increased the infarct volume after
MCAO. The NaHS-induced increase in infarct volume was abolished by the administration of dizolcilpine maleate (an
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N-methyl-D-aspartate receptor channel blocker). MCAO caused an increase in H2S level in the lesioned cortex as well
as an increase in the H2S synthesizing activity. Administration of 4 different inhibitors of H2S synthesis reduced
MCAO-induced infarct volume dose dependently. The potency of these inhibitors in effecting neuroprotection in vivo
appeared to parallel their potency as inhibitors of H2S synthesis in vitro. It also appeared that most of the H2S
synthesizing activity in the cortex results from the action of cystathionine -synthase.
ConclusionsThe present results strongly suggest that H2S plays a part in cerebral ischemic damage after stroke.
Inhibition of H2S synthesis should be investigated for its potential as a novel neuroprotective stroke therapy. (Stroke.
2006;37:889-893.)
Key Words: hydrogen sulfide N-methyl-D-aspartate receptor stroke

I n addition to being a risk factor of cardiovascular diseases,

elevated plasma homocysteine (Hcy) is strongly linked with
increased risk of acute stroke.1,2 Hcy may be converted by the
action of cystathionine -synthase (CBS; EC 4.2.1.22) to cysta-
thionine, which, in turn, is acted on by cystathionine -lyase
(CSE; EC 4.4.1.1) to form cysteine (Cys), thus suggesting a
possible association of the actions of Cys and Hcy in stroke.
Plasma Cys has been reported to be as important a risk factor as
Hcy in coronary heart disease.3 However, it has also been
reported that plasma Cys remained unchanged in stroke patients
with hyperhomocysteinemia.4
Cys has been demonstrated to cause neuronal death when
given orally to infant mice5 and is also an important mediator
of the pathology of brain injury in immature animals sub-
jected to hypoxic-ischemic brain injury.6 In rat hippocampal
slices, Cys appears to be innocuous under normal conditions
but causes toxicity to neurons deprived of glucose, oxygen, or
both.7 Extracellular levels of Cys are also markedly elevated
after ischemic brain injury caused by carotid artery ligation in
Mongolian gerbils.8 Thus, elevation in extracellular Cys may
occur during brain ischemia and contribute to the pathophys-
iology of ischemic brain injury. Cys is not an agonist at the
N-methyl-D-aspartate (NMDA) receptor but is known to cause
neuronal cell death that can be prevented by NMDA antago-
nists.9,10 In this respect, it has been suggested that Cys exerts an
indirect action on the NMDA receptors via the excitatory amino
acids or Cys derivatives such as S-nitrosocysteine or Cys
sulfinate.9,11
However, it is now known that Cys is also the precursor of
the novel gaseous mediator hydrogen sulfide (H2S).12 It is
interesting to note that the 2 enzymes involved in the
conversion of Hcy to Cys are also the major enzymes
involved in the production of H2S from Cys, namely CBS and
CSE.13,14 Both enzymes use Cys as substrate to form H2S with
the byproduct serine, in the case of CBS, or pyruvate and
ammonia, in the case of CSE. The actions of H2S include, in
the cardiovascular system, vasodilation and thus lowering of
blood pressure, and in the brain, the enhancement of NMDA
receptor-mediated responses such as the facilitation of the
induction of hippocampal long-term potentiation.15 The level

Received November 14, 2005; accepted November 30, 2005.

From the Departments of Pharmacology (K.Q., C.P.L.H.C., P.K.M., P.T.-H.W.) and Biochemistry (B.H.), Yong Loo Lin School of Medicine, National
University of Singapore, Kent Ridge, Singapore; and Department of Neurology (C.P.L.H.C.), National Neuroscience Institute, Singapore General Hospital
Campus, Singapore General Hospital.
Correspondence to Dr Peter T.-H. Wong, Department of Pharmacology, National University of Singapore, 18 Medical Dr, Singapore 117597. E-mail
phcwth@nus.edu.sg
2006 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org DOI: 10.1161/01.STR.0000204184.34946.41

889
 890 Stroke March 2006
of endogenous H2S in the brain has been estimated at 50
to 160 mol/L,16,17 concentrations high enough to suggest
physiological functions. Most recently, we observed that high
plasma Cys/cystine levels are associated with poor clinical
outcome in acute stroke patients and that Cys neurotoxicity in
cerebral ischemia in the rat is attenuated by inhibition of
CBS.18 We therefore investigated the effects of H2S and the
inhibition of its formation in a rat model of stroke.
Materials and Methods
Animals and Drugs
Male Wistar rats (200 to 250 g) obtained from the University Laboratory
Animal Center, National University of Singapore were housed singly
under natural light/dark (12 to 12 hours) cycle with food and water
available ad libitum. All procedures were performed in accordance
with institutional guidelines, and all efforts were made to minimize
suffering and the number of rats used.
Cys, DLpropargylglycine (PAG), aminooxyacetic acid (AOAA),
sodium hydrosulfide (NaHS), hydroxylamine (HA), -cyanoalanine
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(-CNA), and dizolcilpine maleate (MK-801) were obtained from
Sigma. All drugs were dissolved in saline and administered by
intraperitoneal injection. Control rats received saline only.
Focal Cerebral Ischemic Stroke Model
Cerebral ischemia was induced by permanent unilateral occlusion of
the left middle cerebral artery (MCA) using a modified subtemporal
approach described previously.19 Briefly, the MCA was occluded
proximal to the origin of the lenticulo-striate branches extending to
the point where it intersects the inferior cerebral vein by electrocau-
terizaton. Rectal temperature was maintained at 370.5C until
recovery from anesthesia. Control rats were sham operated by
omitting only the occlusion.
Measurement of Infarct Volume
Rats were decapitated 24 hours after MCA occlusion (MCAO).
Coronal sections (2 mm) of the cerebrum were then stained with
0.1% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37C for
30 minutes and fixed using 4% formaldehyde in PBS. The size of the
unstained infarcted area per section was analyzed (Olympus Micro
Image Lite 4.0 system), and the true infarct volume was obtained by
correcting for edema.20
H2S Synthesizing Activity in Cortical Homogenate
H2S production in cortical homogenates was studied as described by
Abe and Kimura12 with modification. Rat cerebral cortex was
homogenized in ice-cold 50 mmol/L potassium phosphate buffer, pH
8.0 (12% wt/vol), with a Polytron homogenizer. Homogenate (0.9
mL), preincubated at 37C with or without an inhibitor for 5 minutes
in a 20-mL glass vial, was then cooled on ice for 10 minutes before
L-Cys (10 mmol/L final concentration) and pyridoxal 5-phosphate
(2 mmol/L) were added. The final volume was 1 mL. A 2-mL tube
containing a piece of filter paper (0.51.5 cm) soaked with zinc
acetate (1%; 0.3 mL) was put inside the vial. The vial was then
flushed with a slow stream of nitrogen gas for 20 seconds and then
capped with an airtight serum cap. The vials were then transferred to
a 37C shaking water bath. After 90 minutes, trichloroacetic acid
(TCA; 50%; 0.5 mL) was injected into the reaction mixture through
the serum cap. Another 60 minutes was allowed for the trapping of
evolved H2S by the Zn acetate solution as Zn sulfide. Then the serum
cap was removed and N, N-dimethyl-p-phenylenediamine sulfate
(20 mmol/L; 50 L) in 7.2 mol/L HCl and FeCl3 (30 mmol/L; 50
L) in 1.2 mol/L HCl were added to the inner tube. After 20 minutes,
absorbance at 670 nm was measured with a microplate reader
(Sunrise; TECAN).
The calibration curve of absorbance versus H2S concentration was
obtained by using NaHS solution of varying concentrations. When
NaHS is dissolved in water, HS is released and forms H2S with H.
H2S concentration was taken as 30% of the NaHS concentration in
the calculation.22 The calibration curve was linear from 0 to
320 mol/L NaHS or 96 mol/L H2S.
Measurement of Endogenous Level of H2S
Rats were killed and the head was flash-frozen with liquid nitrogen.
Cortical tissues were removed without thawing and then homoge-
nized in ice-cold 50 mmol/L potassium phosphate buffer, pH 8.0
(12% wt/vol), with a Polytron homogenizer. The homogenate was
centrifuged (47 000g; 10 minutes; 4C) and the supernatant (75 L)
was mixed with 0.25 mL Zn acetate (1%) and 0.45 mL water for 10
minutes at room temperature. TCA (10%; 0.25 mL) was then added,
centrifuged (14 000g; 10 minutes; 4C), and the clear supernatant
was collected and mixed with N,N-dimethyl-p-phenylenediamine
sulfate (20 mmol/L; 133 L) in 7.2 mol/L HCl and FeCl3
(30 mmol/L; 133 L) in 1.2 mol/L HCl. After 20 minutes, absor-
bance was measured as described above.
Reverse TranscriptasePolymerase Chain Reaction
Total mRNA from brain tissues were extracted using TriZol reagent
(Invitrogen) as described.19 RNA (10 g) was reverse transcribed
into cDNA using Avian Myeloblastosis Virus reverse transcriptase
(Promega) and oligo dT12 as primers. The sequence of primers,
annealing temperature/time, and PCR cycle are as follows: -actin
(internal standard; 870 bp; 60C/2 minutes; 29 cycles); sense:
5-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3; anti-
sense: 5-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3;
CBS (559 bp; 58C/2 minutes; 32 cycles); sense: 5-ATGCTGCAG-
AAAGGCTTCAT-3; antisense: 5-GTGGAAACCAGTCGGTGT-
CT-3; CSE (579 bp; 58C/2 minutes; 32 cycles); sense: 5-CGCA-
CAAATTGTCCACAAAC-3; antisense: 5-GCTCTGTCCTTCT-
CAGGCAC-3.
PCR amplification products were analyzed on a 1% agarose gel.
Bands were visualized with an ultraviolet transilluminator and band
intensities were quantified by a SynGene Multi Genius Bio Imaging
System.
Statistical Analysis
All comparisons were performed by 1-way ANOVA followed by
post hoc analysis with Bonferroni correction using SPSS for Win-
dows (v13). Data are expressed as meanSEM. The critical P level
set for significance is 0.05.
Figure 1. Dose-dependent effect of NaHS on infarct volume 24
hours after MCAO. NaHS (0.18 mmol/kg or otherwise stated in
parentheses) was injected intraperitoneally 10 minutes before
MCAO. MK-801 (3 mol/kg), injected intraperitoneally 10 min-
utes before NaHS administration, completely abolished the
effect of NaHS. The mean infarct volume was 17910 mm3
(100%) for control rats receiving saline only. n4. One-way
ANOVA: F(3,12)7.389; P0.01. *P0.01 against control group
by post hoc analysis with Bonferroni correction.

Figure 2. H2S levels in cortical tissues 24 hours after MCAO
with or without Cys loading. Cys (10 mmol/kg) was injected in-
traperitoneally 50 minutes before MCAO. n4. One-way
ANOVA: F(2,9)52.917; P0.001. *P0.001 against the sham-
operated control group and **P0.02 against MCAO group by
post hoc analysis with Bonferroni correction.
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Results
MCAO causes widespread tissue infarction predominantly in
the cortex of the occluded side of the brain. Administration of
NaHS at 0.09 mmol/kg (IP) before MCAO had no significant
effect on the infarct volume, but at 0.18 mmol/kg, the infarct
volume was increased to 150% of control. NaHS was
administered at a sublethal dose,17 and no increase in mortality
was observed in this group of rats. Coadministration of the
NMDA receptor channel blocker MK-801 (3 mol/kg) com-
pletely abolished the proinfarct effect of NaHS (Figure 1).
The endogenous level of H2S in the cerebral cortex almost
doubled in the lesioned cortex after MCAO when compared
with sham-operated controls. As expected, H2S levels in-
creased further after previous Cys loading (Figure 2). In
addition, the H2S synthesizing activity, at maximal Cys
concentration, in cortical homogenates also increased after
MCAO (Figure 3). However, when the cortical expression of
CBS and CSE were investigated using RT-PCR, no signifi-
cant difference in the expression of either enzyme was
observed between sham-operated and MCAO rats (Figure 4).
It is noted that the expression of CSE was observed to be
higher than that of CBS in both groups of animals.
Figure 3. H2S synthesizing activity in cortical homogenate 24
hours after MCAO. H2S assay was performed as described in
the Methods. n4. *P0.05 against sham-operated control rats
by independent sample t test; t2.970; df6.
Qu et al Hydrogen Sulfide and Stroke 891
Figure 4. Cortical expression of CBS and CSE mRNA in sham-
operated and MCAO rats. RT-PCR was performed as described
in the Methods. No significant difference in expression of either
enzyme was detected between sham-operated and MCAO rats
(independent sample t test). n3. Inset, Representative bands
for CBS, CSE, and -actin RT-PCR products obtained by gel
electrophoresis.
To gain further insight into the role played by H2S in this
stroke model, the inhibitory effects of 2 CBS inhibitors
(AOAA and HA) and 2 CSE inhibitors (-CNA and PAG) on
the H2S synthesizing activity in cortical homogenate was
studied. All 4 inhibitors inhibited H2S production in vitro in
a dose-dependent manner (Figure 5). AOAA exhibited the
greatest potency with an IC50 value of 12.6 mol/L, reaching
98% inhibition at a concentration of 0.5 mmol/L (data not
shown in Figure 5). The IC50 values for the other inhibitors
are 0.5 mmol/L (HA), 2.5 mmol/L (-CNA), and 7.1 mmol/L
(PAG). In addition, the CSE inhibitors achieved only 70%
(-CNA) and 55% (PAG) inhibition at the highest concen-
tration used (10 mmol/L).
Administration of each of the 4 inhibitors to rats before
MCAO revealed that all 4 compounds were able to reduce
infarct volume in a dose-dependent manner (Figure 6).
Notably, their effectiveness in this respect paralleled their
ability to inhibit H2S synthesis in vitro, thus AOAA was the
most effective at a dose of 0.05 mmol/kg followed by HA,
Figure 5. Inhibition of H2S synthesizing activity in cortical ho-
mogenate by AOAA (), HA (), -CNA () and PAG (). The
H2S assay was performed as described in the Methods. Each
point represents the meanSEM of 3 independent experiments
determined in duplicate. The calculated IC50 values were
12.6 mol/L (AOAA), 0.5 mmol/L (HA), 2.5 mmol/L (-CNA),
and 7.1 mmol/L (PAG).
 892 Stroke March 2006
Figure 6. Dose-dependent effect of AOAA, HA, -CNA, and
PAG on infarct volume 24 hours after MCAO. Inhibitors were
injected intraperitoneally at various doses 50 minutes before
MCAO. The mean infarct volume was 19712 mm3 (100%) for
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the control rats receiving saline only. n5 to 6. One-way
ANOVA for AOAA: F(4,20)7.389, P0.01; HA: F(3,16)27.532,
P0.001; -CNA: F(3,18)5.753, P0.01; PAG: F(3,16)41.150,
P0.001. *P0.05 and **P0.01 against control group by post
hoc analysis with Bonferroni correction.
which was effective at 0.5 to 1.0 mmol/kg. It is interesting to
note that AOAA was not effective at higher doses (ie, up to
0.5 mmol/kg). In contrast, -CNA and PAG were effective at
much higher doses of 1 or 2 mmol/kg. However, relative to
-CNA, PAG appeared to be more effective in reducing
infarct volume than its potency in inhibiting H2S production
suggested (Figure 5).
Discussion
Cys has been shown previously to increase infarct volume
after MCAO in a dose-dependent manner.18 This effect of
Cys was abolished by AOAA, a CBS inhibitor, suggesting
that Cys exerts its effect after conversion to H2S via the action
of CBS in the brain. We show here that NaHS, an H2S donor,
is also able to enhance the destructive effects of cerebral
ischemia, leading to a marked increase in the extent of tissue
damage. At a dose of 0.18 mmol/kg of NaHS, the effective
dose of H2S is 0.06 mmol/kg based on a yield of 30%;21
this is equivalent to only 0.6% of the effective dose of Cys at
10 mmol/kg. This is therefore consistent with the possibility
that Cys increased the cerebral infarct by production of H2S.
Moreover, the effects of both Cys (K. Qu and P.T.H. Wong,
unpublished data, 2005) and NaHS (Figure 1) were abolished
by MK-801 pretreatment, confirming that H2S acts most
likely by an effect via NMDA receptors.
It has been reported previously that physiological concen-
trations of H2S enhances NMDA receptor function through
activation of adenylyl cyclase. Increased production of
cAMP, observed in primary cultures of both neuronal and
glial cells, may lead to phosphorylation of the NMDA
receptor subunits at specific sites by protein kinase A,
resulting in the activation of NMDA receptor-mediated exci-
tatory postsynaptic current.22 Thus, in cerebral ischemia, H2S
may enhance the NMDA receptor mediated excitotoxicity of
glutamate. Together with the observed increase in the endog-
enous level of both H2S and H2S synthesizing activity in the
MCAO lesioned cortex (Figures 2 and 3), these various
observations strongly suggest that H2S plays an important
role in tissue damage in the ischemic brain, possibly through
enhancement of NMDA receptor-mediated calcium overload.
Another possibility is that H2S influences the ischemic
infarction by altering cerebral blood flow. However, because
H2S causes vasodilation and vasodilators are generally cere-
broprotective, leading to reduced infarct size;23 this possible
mechanism is much less likely.
The ability of cortical tissue to increase production of H2S
very quickly after MCAO or Cys loading suggests that the
enzyme responsible for this conversion is not saturated by its
substrate in vivo. Abe and Kimura12 noted that CBS inhibitors
including AOAA completely inhibited the production of H2S
in rat whole brain homogenates, whereas CSE inhibitors,
including PAG, were ineffective at a concentration of
2 mmol/L. Abe and Kimura12 concluded that CBS is the
predominant enzyme responsible for H2S production in the
brain. It has also been reported that CBS is localized in most
areas of the adult mouse brain but predominantly in the cell
bodies and neuronal processes of Purkinje cells and Ammons
horn neurons.24
Consistently, our present results (Figure 5) also suggest a
predominantly CBS-catalyzed production of H2S in the cere-
bral cortex. AOAA inhibited H2S production effectively with
an IC50 value of 12.6 mol/L and caused almost complete
inhibition of H2S production at 0.5 mmol/L. In contrast, PAG,
a potent CSE inhibitor, inhibited cortical H2S production with
an IC50 value of 7.1 mmol/L, suggesting that PAG may be
acting as a low-affinity inhibitor of CBS in this instance
rather than as an inhibitor of CSE. It has been reported that
CSE is not expressed at detectable levels or expressed at a
barely detectable level in the rat and mouse brain by Northern
or Western blot analysis.12 However, it has to be noted that
the expression of CSE mRNA (by RT-PCR) is apparently
higher than that of CBS (Figure 4), which is in stark contrast
to the data obtained in the in vitro assay (Figure 6). It is
possible that CSE is expressed at the mRNA level but not at
the protein level. More conclusive studies can be made only
when antibodies to both CBS and CSE become available.
All 4 inhibitors used reduced the MCAO-induced infarct
volume in a dose-dependent manner. The rank order of
potency was AOAAHAPAG-CNA (Figure 6).
Significantly, the observed potencies of the compounds as
H2S synthesis inhibitors in vitro paralleled their effectiveness
in reducing MCAO infarct size in vivo. AOAA, as the most
potent inhibitor, significantly reduced infarct volume at a
dose of 0.05 mmol/kg. Interestingly, at higher doses, AOAA
no longer exhibited any protective effects, probably indicat-
ing overinhibition of H2S formation, leading to detrimental
effects, supporting an important neuromodulator role for H2S
in the brain. It was further noted that at doses 0.5 mmol/kg,
rats showed an unacceptably high mortality rate (data not
shown).
Summary
The present results clearly demonstrate that H2S, produced
from Cys in the cerebral cortex most probably by CBS, is an
important mediator of ischemic damage. H2S acts via the

NMDA receptor, which has become a prime target for stroke
research over the past decade. Indeed, some NMDA antago-
nist and glycine antagonists have shown promise in clinical
trials.25,26 Current evidence suggests that H2S promotes ische-
mic damage by a direct degenerative effect on cerebral
neurons, although effect on cerebral blood flow may not be,
as yet, excluded. Whatever the mechanism of action, these
results suggest, for the first time, that inhibition of H2S
production using a CBS inhibitor may represent a novel
therapeutic approach to the treatment of stroke.
Acknowledgments
This work was supported by a Biomedical Research Council of
Singapore grant awarded to P.T.H.W. (PI), C.P.L.H.C., and B.H.
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 Hydrogen Sulfide Is a Mediator of Cerebral Ischemic Damage
Kun Qu, Christopher P.L.H. Chen, Barry Halliwell, Philip K. Moore and Peter T.-H. Wong

Stroke. 2006;37:889-893; originally published online January 26, 2006;

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doi: 10.1161/01.STR.0000204184.34946.41
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Copyright 2006 American Heart Association, Inc. All rights reserved.
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