The administration of hydrogen sulphide prior to ischemic reperfusion has neuroprotective effects in an acute stroke model
The administration of hydrogen sulphide prior to ischemic reperfusion has neuroprotective effects in an acute stroke model
Chul-Woong Woo 0 1 2
Jae-Im Kwon 0 1 2
Kyung-Won Kim 0 2
Jeong-Kon Kim 0 2
Sang- Beom Jeon 0 2
Seung-Chae Jung 0 2
Choong-Gon Choi 0 2
Sang-Tae Kim 0 1 2
Jinil Kim 0 1 2
Su Jeong Ham 0 1 2
Woo-Hyun Shim 0 1 2
Yu Sub Sung 0 2
Hyun Kwon Ha 0 2
Yoonseok Choi 0 2
Dong- Cheol Woo 0 1 2
0 Institute for Life Sciences, Asan Medical Center , grant number 2015-651 to HKH (
1 Asan Institute for Life Sciences, Asan Medical Center , Songpa-gu, Seoul , Republic of Korea, 2 Department of Radiology, Asan Medical Center , Songpa-gu, Seoul , Republic of Korea, 3 Department of Neurology, Asan Medical Center , Songpa-gu, Seoul , Republic of Korea, 4 Medical Research Institute, Gangneung Asan Hospital Gangneung-si , Gangwon-do , Republic of Korea
2 Editor: Quan Jiang, Henry Ford Health System , UNITED STATES
Emerging evidence has suggested that hydrogen sulfide (H2S) may alleviate the cellular damage associated with cerebral ischemia/reperfusion (I/R) injury. In this study, we assessed using 1H-magnetic resonance imaging/magnetic resonance spectroscopy (1H-MRI/MRS) and histologic analysis whether H2S administration prior to reperfusion has neuroprotective effects. We also evaluated for differences in the effects of H2S treatment at 2 time points. 1H-MRI/ MRS data were obtained at baseline, and at 3, 9, and 24 h after ischemia from 4 groups: sham, control (I/R injury), sodium hydrosulfide (NaHS)-30 and NaHS-1 (NaHS delivery at 30 and 1 min before reperfusion, respectively). The total infarct volume and the midline shift at 24 h post-ischemia were lowest in the NaHS-1, followed by the NaHS-30 and control groups. Peri-infarct volume was significantly lower in the NaHS-1 compared to NaHS-30 and control animals. The relative apparent diffusion coefficient (ADC) in the peri-infarct region showed that the NaHS-1 group had significantly lower values compared to the NaHS-30 and control animals and that NaHS-1 rats showed significantly higher relative T2 values in the peri-infarct region compared to the controls. The relative ADC value, relative T2 value, levels of N-acetylL-aspartate (NAA), and the NAA, glutamate, and taurine combination score (NGT) in the ischemic core region at 24 h post-ischemia did not differ significantly between the 2 NaHS groups and the control except that the NAA and NGT values were higher in the peri-infarct region of the NaHS-1 animals at 9 h post-ischemia. In the ischemic core and peri-infarct regions, the apoptosis rate was lowest in the NaHS-1 group, followed by the NaHS-30 and control groups. Our results suggest that H2S treatment has neuroprotective effects on the peri-infarct region during the evolution of I/R injury. Furthermore, our findings indicate that the administration of H2S immediately prior to reperfusion produces the highest neuroprotective effects.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
kr). The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Blood flow reperfusion is considered to be the most important intervention to preserve
neurologic function when managing patients with acute cerebral stroke. The infusion of
thrombolytics such as recombinant tissue-type plasminogen activator (rt-PA) either intravenously (i.v.)
or through endovascular catheterization are standard options to manage reperfusion [
However, the reperfusion of ischemic tissue is often associated with tissue damage due to
various factors such as inflammatory responses, oxidative stress, and excitotoxicity generated by
excessive glutamate release .Therefore, many research groups have attempted to develop
novel methods for reducing cerebral ischemic/reperfusion (I/R) injury [3±5].
Hydrogen sulfide (H2S) has for many decades been primarily considered a pungent toxic
gas and an environmental hazard [
]. A number of studies on H2S have reported that it has a
broad range of physiological and pathophysiological functions, including the regulation of
neuronal activity, induction of angiogenesis, and vascular relaxation [7±10]. Over the previous
decade, there have also been several reports that H2S exerts neuroprotective effects in animal
models of cerebral I/R injury, by inhibiting oxidative stress, inflammation, and apoptosis [11±
15]. However, only a limited number of studies have involved the administration of H2S prior
to reperfusion. Furthermore, there have been no reports on the optimal administration time of
H2S in a cerebral stroke model which is a major point of consideration.
In our current study, we aimed to evaluate the neuroprotective effects of H2S when
administered prior to reperfusion in a rat cerebral acute stroke model, and also investigate whether
the timing of H2S treatment influences its neuroprotective effects via a systematic analysis of
temporal evolution data generated using vivo 1H-MRI/MRS and histology.
Materials and methods
This study was carried out in strict accordance with the guidelines of the National Institutes of
Health. All of the procedures were performed following approval by the Institutional Animal
Care and Use Committee of the Asan Medical Center (IACUC Number: 2015-14-157). All
surgery was performed under isoflurane anesthesia. All the animals were carefully monitored by
the trained individuals who can assess the animal pain behavior. Animal euthanasia was
planned if the animal showed continuous pain related behavior; however, no animal showed
the unrelieved severe pain related behavior during the experimental period.
Transient middle cerebral artery occlusion (tMCAO) model
We used male Sprague-Dawley rats (eight weeks old, n = 48; weight = 280±310 g; Orient Bio,
Pyeongtaek, Republic of Korea). All animals were individually housed in standard plastic cages
and maintained on a 12-h light-dark cycle (lights on at 08:00 A.M.) at an ambient temperature
of 24.0±25.0ÊC, with free access to food and water.
The transient middle cerebral ischemic occlusion (tMCAO) model was used to generate
I/R injury in rats. Rats were initially anaesthetised with 5% isoflurane and maintained with
2±3% isoflurane during surgery. Middle cerebral artery (MCA) occlusion, using a previously
described method of intraluminal vascular occlusion [
], was performed for 60 min to induce
ischemia. The MCA occlusion was then relieved to induce reperfusion. In particular, an 18±
20-mm 4±0 suture thread (Ethylon surgical monofilament polyamide; Ethicon, Livingston,
UK) with a fire-polished tip (diameter, 0.38±0.40 mm) was advanced from the external carotid
artery into the lumen of the internal carotid artery until it blocked the origin of the MCA.
After 60 min, the inserted intravascular thread was removed gently.
2 / 16
To identify animals wherein the tMCAO model was successfully established, the regional
cerebral blood flow (CBF) was monitored before and after MCA occlusion by using a laser
Doppler flow (LDF) monitoring device (VMS-LDF, Moor Instruments, Devon, UK). For the
placement of the LDF probe, a burr hole (diameter, 2 mm) was drilled 2 mm posterior and 6
mm lateral to the bregma, with care being taken not to injure the underlying dura mater [
Rats that did not show a significant CBF reduction after MCA occlusion (at least 70% decrease
from the baseline value) were excluded from the experimental group [
]. Sham-operated rats
were manipulated in the same way without MCA occlusion.
Sodium hydrosulfide (NaHS, Sigma-Aldrich, St. Louis, MO) dissolved in saline (25 μmol/kg of
NaHS dissolved in 2.5 ml of saline) was used as an H2S donor. Normal saline (2.5 ml) was used
for the vehicle. The drug and vehicle were administrated via intravenous injection.
Rats were randomly divided into 4 groups (n = 8 per group): (1) sham-operated group with
no I/R modelling or injection of the vehicle (n = 8), (2) control group with I/R modelling and
injection of the vehicle (n = 8), (3) NaHS-30 group with I/R modelling and injection of the
drug at 30 min before reperfusion (n = 8), and (4) NaHS-1 group with I/R modelling and
injection of the drug at 1 min before reperfusion (n = 8). If death or insufficient infarction
occurred, additional rats were included to meet the sample size number.
MRI and 1H-MRS
1H-MRI/MRS was obtained at baseline (i.e. before I/R modelling) and at 3, 9, and 24 h after
I/R modelling (Fig 1). MRI and 1H-MRS were conducted using a 9.4T/160 mm animal MR
system (Agilent Technologies, Santa Clara, CA). A 72-mm birdcage volume coil was used for
excitation, and a 4-channel phased array surface coil served as the receiving coil. All the
animals were anaesthetised through a mask via the spontaneous inhalation of 2.0±2.5% isoflurane
Fig 1. Summary of NaHS administration and MRI/MRS acquisition times in the experimental groups.
3 / 16
in a 1:2 mixture of O2:N2O. Respiration was monitored and rats were maintained in a
normothermic condition at 37.5 ± 0.5ÊC using an air heater system.
The MRI protocol included T2-weighted images (T2-WIs), T2 maps, and spin-echo
diffusion weighted images (DWIs). T2-WIs were acquired with a fast spin-echo sequence (TR, 4000
ms; k-zero, 3; echo spacing, 10.98 ms; 32 segments; echo train length, 8; effective TE, 32.95 ms;
averages, 1; matrix, 256 × 256; field of view, 30 × 30 mm; and slice thickness, 1.0 mm, no gap).
T2 map images were acquired using a multi-echo multi-slice (MEMS) sequence with the
following parameters: TR, 3000 ms; TE, 10±150 ms; 15 echoes; averages, 1; matrix, 128 × 128; and
slice thickness, 1 mm, no gap. Furthermore, the DWI parameters were as follows: TR, 2000
ms; TE, 22.67 ms; averages, 1; matrix, 128 × 128; slice thickness, 1 mm, no gap; and b-values, 0
and 1000 s/mm2. Quantitative ADC maps were created on a voxel-wise basis, with a linear
least-squares fit on the logarithm of the signal intensity vs. the b-value for each diffusion
direction. The geometrical imaging parameters (i.e., number and orientation of slices, FOV) of the
T2 maps and DWIs were the same as those used on T2-WIs.
1H-MRS was performed to detect the metabolites in vivo and monitor the temporal changes
caused by the stroke. In particular, the N-acetyl-L-aspartate (NAA) concentration and the
combination score of NAA, glutamate (Glu), and taurine (Tau) (NAA + Glu + Tau, NGT)
have been proposed as markers of neuronal density and viability in stroke [
]. The MR
spectra of standard brain metabolites were collected from a single voxel of 3.5 × 2 × 1.6 mm3
in the ischemic core (lateral caudo-putamen and somato-sensory cortex) and peri-infarct
region (primary motor cortex and somato-sensory cortex) in the slice, according to the
diagram suggested previously by Zhao et al  (Fig 2). For single voxel localisation of 1H-MRS
images, we used point resolved spectroscopy sequence (PRESS) with the variable power RF
pulses with optimised relaxation delays (VAPOR) method (TR/TE, 5000/13.47 ms; spectral
width, 5 kHz; number of averages, 256; data points, 2048). Respiration gating was used for
DWI scan acquisitions, and the total scan time was <90 min.
All MRI data were assessed by an observer blinded to the grouping information. MRI analysis
was conducted using ImageJ software (National Institutes of Health, Bethesda, Maryland;
http://rsbweb.nih.gov/ij/). The total infarct volume at 24 h after ischemia was measured on
T2-WI scans using the 2D volumetry technique, which involves the summation of the infarct
Fig 2. Diagram of the regions of the ischemic core and the peri-infarct region.
4 / 16
volume measured from each slice [
]. The peri-infarct volume at 24 h after ischemia was also
measured on T2-WI scans, which involves the infarct volume measured from the peri-infarct
region in the slice (Fig 2).The midline shift (MLS) quantification method was used to
determine the space-occupying effect of the cerebral edema [
]. This method was performed on
T2-WI scans at 24 h after ischemia, where the position of the third ventricle could be
determined clearly in all animals. The distance between the outer border of the cortex and the
middle of the third ventricle was measured from the ipsilateral (A) and contralateral (B) sides (Fig
3). Measurements were obtained at the level of the maximum lateral displacement of the
ventricle. MLS was calculated using the following equation: MLS = (A − B)/2, as described
The degree of ischemic injury was evaluated by measuring the ADC and T2 values on a
respective map. The regions-of-interest (ROIs) were located in the ischemic core and
periinfarct region of the ipsilateral hemisphere, and also in the corresponding ROIs in the
Fig 3. Infarct volumes and midline shift (MLS). (A) The peri-infarct volume was measured from the peri-infarct region (yellow region) on T2-WIs at 24 h
after ischemia in representative rats from each group. To calculate the MLS, the distance between the outer border of the cortex and the middle of the third
ventricle was measured from the ipsilateral (red line) and contralateral (blue line) sides. (B -D) The total infarct volume (B), peri-infarct volume (C) and MLS
(D) were lowest in the NaHS-1 group, followed by the NaHS-30 and control groups. The peri-infarct volume was significantly lower in the NaHS-1 group than
in the NaHS-30 group. Data are presented as mean ± standard deviation (n = 8 rats in each group). *P < 0.001 vs. the control group, # P < 0.001 vs. the
5 / 16
contralateral hemisphere (Figs 4 and 5). Thereafter, the relative ADC (rADC) and T2 (rT2)
values were calculated as ratios i.e., ipsilateral value/contralateral value, as previously decribed [
The resulting spectra were processed as described by Lei et al [
]. Briefly, absolute
quantification was obtained using a linear combination analysis method (LC Model ver.6.0, Los Angeles,
CA). The MR spectra were considered acceptable if the signal-to-noise ratio (SNR) was
and the standard deviation (CrameÂr-Rao lower bounds, CRLB) of the spectral fit for the
metabolite was <30%. The concentrations of NAA (cNAA) and NGT (cNGT) were measured
in the ischemic core and peri-infarct regions at baseline and at 3, 9, and 24 h after ischemia.
Terminal transferase d-UTP nick-end labelling (TUNEL) assays
The extent of apoptosis in the damaged tissues was assessed using the TUNEL assay. For this
purpose, we used the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon, CA).
Fig 4. Effect of NaHS treatment on the ADC value. (A) The mean ADC values were measured in the ipsilateral (ischemic core [white square] and
peri-infarct [red square]) and contralateral regions on the ADC maps at baseline and at 3, 9, and 24 h after ischemia. (B, C) The rADC of the ischemic
(B) core and (C) peri-infarct regions were plotted against time. Data are presented as a mean ± standard deviation (n = 8 rats in each group). *
P < 0.001 vs. the control group, # P < 0.001 vs. the NaHS-30 group.
6 / 16
Fig 5. Effect of NaHS treatment on the T2 value. (A) The mean T2 values were measured in the ipsilateral (ischemic core [white square] and peri-infarct
[red square]) and contralateral regions on serial T2 maps at baseline and at 3, 9, and 24 h after ischemia. (B, C) The rT2 of the ischemic (B) core and (C)
peri-infarct regions were plotted against time. Data are presented as mean ± standard deviation (n = 8 rats in each group). * P < 0.001 vs. the control group,
# P < 0.001 vs. the NaHS-30 group.
The rats were sacrificed and fixed by cardiac perfusion with 4% paraformaldehyde at 24 h
after I/R modelling. The brain tissue was isolated and fixed in 4% paraformaldehyde for 3
days. The fixed brain tissues were then sectioned coronally (thickness, 3 μm) and mounted on
prechilled glass slides coated with poly-L-Iysine. Tissue sections were incubated in a dry oven
for 1 h at 60ÊC. The tissues were treated with a working solution containing reaction buffer
and enzyme (7:3) mixture for 1 h at 37ÊC, followed by an anti-digoxigenine peroxidase
conjugate for 30 min at room temperature. The tissues were then treated with the DAB substrate
(1:50) for 5 min in a dark room, followed by haematoxylin for 2 minutes in the dark room for
The TUNEL-stained sections were examined under a microscope (ZEISS, HAL100, 200×
magnification) and photographed. A brown stain in the nucleus represents an apoptotic cell.
The number of TUNEL-positive cells and total cells were counted using ImageJ software in 3
7 / 16
randomly chosen fields from the ischemic core and peri-infarct regions. The percentage of
TUNEL-positive cells relative to the total cell count was used to evaluate the apoptosis rate.
All data are expressed as means ± standard deviation. Statistical analysis was performed using
SPSS version 13.0 software (SPSS, Chicago, IL). The CBF reduction, total infarct volume, MLS,
rADC, rT2, cNAA, cNGT, and apoptosis rate in multiple groups were compared using
oneway analysis of variance with multiple post-hoc comparison with Scheffe's method. Differences
with a P value <0.05 were considered statistically significant.
The success rate of tMCAO modelling was 60% (24 included animals, with 40 receiving
surgery). The causes for exclusion were as follows: death during operation [control (n = 2) and
NaHS-1 (n = 2) groups], death after operation [control (n = 4), NaHS-1 (n = 1), and NaHS-30
(n = 3) groups], and no infarction or insufficient infarction generated [NaHS-1 (n = 2) and
NaHS-30 (n = 2) groups]. The 4 rats without any infarction were excluded after laser Doppler
monitoring indicated insufficient CBF reduction, or after MRI performed at 3, 9, and 24 h
after I/R modelling did not show any signs of infarction. All the included rats in the NaHS-1,
NaHS-30, and control groups exhibited marked decreases in the regional CBF after ischemia
i.e., > 70% reduction, compared to the baseline regional CBF (Table 1). The rats in the
shamoperated group did not show any reduction in the CBF. There was no significant difference in
CBF reduction among the NaHS-1, NaHS-30, and control groups (81% ± 5%, 81% ± 5%, and
79% ± 5%, respectively, P = 0.9019, one-way ANOVA).
Infarct volumes and MLS on T2-WI scans
Fig 3 shows the infarct lesions that were evident on T2-WI scans at 24 hours after I/R
modelling in the representative rats from each group. The total infarct volume, peri-infarct volume,
and MLS at 24 h after I/R modelling were lowest in the NaHS-1 group, followed by the
NaHS30 and control groups, which is indicative of the neuroprotective effect of NaHS. In the total
infarct volume and MLS analyses, one-way ANOVA post-hoc tests showed that both NaHS
treatment groups had significantly lower values than the control group (NaHS-30 v.s. Control,
P < 0.001; NaHS-1 v.s. Control, P < 0.001), however, no significant difference was observed
between the NaHS-1 and NaHS-30 groups. In the peri-infarct volume analysis, the results
showed that the NaHS-1 treated group had significantly lower values than either the control or
NaHS-30 treated groups (NaHS-30 vs. NaHS-1, P < 0.001; NaHS-1 vs. Control, P < 0.001).
8 / 16
ADC and T2 values
Figs 4 and 5 show the evolution of the lesions from ADC maps and T2 maps at baseline, and at
3, 9, and 24 h after I/R modelling in the representative rats from each group. The ischemic/
infarcted area in the ipsilateral hemisphere showed low ADC values and high T2 values,
compared to the contralateral hemisphere, thus reflecting the degree of cytotoxic/vasogenic
cerebral oedema. In the sham-operated group, the ADC and T2 values did not differ over time. On
serial ADC maps, the rADC of the ischemic core and peri-infarct region decreased over time
in the control, NaHS-1, and NaHS-30 groups. On serial T2 maps, the rT2 of the ischemic core
and peri-infarct region increased over time in the control, NaHS-1, and NaHS-30 groups. The
rADC and rT2 of the ischemic core reached a similar level at 24 h after I/R modelling, with no
significant difference found among the control, NaHS-1, and NaHS-30 groups (P > 0.05,
oneway ANOVA). In contrast, the rADC and rT2 values from the peri-infarct region differed
significantly between the groups at 24 h after I/R modelling (P < 0.001, one-way ANOVA).
Posthoc tests revealed the highest value in the NaHS-1 group, followed by the NaHS-30 group and
control group, with significant differences between the groups (adjusted P < 0.05 for each
comparison, Scheffe's test). These results suggest that the strongest neuroprotective effects,
particularly those for the preservation of the peri-infarct region (i.e., penumbra), were in the
cNAA and cNGT on 1H-MRS
Figs 6 and 7 show the MR spectra for the ischemic core and peri-infarct region, respectively, at
baseline and at 3, 9, and 24 h after ischemia in representative animals from each group. On
serial 1H-MRS analysis of the ischemic core and peri-infarct regions, the cNAA and cNGT
levels were found to decrease over time in the control, NaHS-1, and NaHS-30 groups, reflecting
the temporal evolution of the metabolites associated with I/R injury. In the ischemic core and
peri-infarct regions, the cNAA and cNGT values did not differ significantly between the
groups at the 3 h and 24 h time points (one-way ANOVA, P > 0.05), although post-hoc tests
indicated that the peri-infarct region of the NaHS-1 animals had significantly higher cNAA
and cNGT values than those of the control and NaHS-30 groups at 9 h.
Extent of apoptosis
Positively stained apoptotic cells (round with brown nuclei) were analyzed in the NaHS-1,
NaHS-30, and control groups (Fig 8). In the ischemic core and peri-infarct regions, the lowest
apoptosis rate was observed in the NaHS-1 group, followed by the NaHS-30 and control
groups, with these differences showing significance (P < 0.001, one-way ANOVA). Post-hoc
tests further revealed that the apoptosis rate was significantly lower in the NaHS treatment
groups than in the control group (P < 0.001). Moreover, a significant difference in the
ischemic core and peri-infarct region apoptosis rates was observed between the NaHS-1 and
NaHS30 groups (P < 0.001).
Consistent with the results of prior studies [
11, 14, 21, 26
], we found in our current analysis
that H2S has therapeutic effects against I/R injury in the brain. Using T2-WI analyses, which is
the best sequence for anatomic evaluations (total infarct volume, peri-infarct volume and
MLS), H2S treated rats showed reduced infarct volumes and MLS values compared with
control animals at 24 h post-ischemia. Moreover, our NaHS-1 treatment group showed
significantly better neuroprotective effects compared with the NaHS-30 and control groups.
9 / 16
Fig 6. cNAA and cNGT changes in the ischemic core. (A) MR spectra at baseline and at 3, 9, and 24 h after ischemia. (B, C) The cNAA (B) and cNGT
(C) values were plotted against time. The cNAA and cNGT levels decreased over time in the control, NaHS-1, and NaHS-30 groups and reached a similar
level at 24 h after I/R modelling in all three groups. The cNAA and cNGT values did not differ significantly between the three groups at any time point.Data
are presented as mean ± standard deviation (n = 8 rats in each group).
Although H2S treated groups showed overall therapeutic effects compared to the control
group, our data showed some discrepancies and conflicts in therapeutic efficacies according to
the analysis methods and the measurement times. We assumed that it arose from the
heterogeneities of pathological progression for each model entity; however, if the refined analysis
technology in the stroke research is introduced rather than the current analysis method, we believe
that this problem can be amended.
Using rADC and rT2 measures that respectively represent the formation of cellular edema
] and vasogenic edema [
], we found from our current analysis that H2S administered at 1
min before reperfusion showed a significant decrease in these values at the peri-infarct sites
which are widely regarded as salvagable (penumbra). With regard to the influence of H2S
administration timing, our results indicated that applying this treatment at 1 min before
reperfusion produced better neuroprotective effects in the peri-infarct region compared with a far
10 / 16
Fig 7. cNAA and cNGT changes in the peri-infarct region. (A) MR spectra at baseline and at 3, 9, and 24 h after ischemia. (B, C) The cNAA (B) and
cNGT (C) values were plotted against time. The cNAA and cNGT levels decreased over time in the control, NaHS-1, and NaHS-30 groups and reached a
similar level at 24 h after I/R modelling in all three groups. The cNAA and cNGT values did not differ significantly between the three groups at any time point
other than 9 h after I/R modelling. Data are presented as mean ± standard deviation (n = 8 rats in each group). * P < 0.001 vs. the control group.
earlier administration (30 min) Consistently, some previous studies that have used a
myocardial infarction model have reported that H2S treatment at the onset of reperfusion led to a
significant decrease in the myocardial infarct size [
]. However, no previous study has
assessed the effect of H2S administration at 1 min before reperfusion in a cerebral I/R model.
We postulated that the pharmacokinetic properties of H2S may underlie these timing
effects. When reperfusion occurs, the blood that is newly returning into the ischemic area
carries the initial abundant inflammatory response and oxidative stress factors. In addition,
previous reports indicate that H2S reaches its highest level within a few minutes after the
]. It thus seems reasonable that exposure to H2S immediately before
reperfusion would have more protective effects against initial reperfusion damage. It must
be noted however that we only examined a single dose of H2S in our analysis (25 μmol/kg,
11 / 16
Fig 8. NaHS treatment suppresses apoptosis. (A) Representative photomicrographs of TUNEL staining results in the ischemic core and peri-infarct regions
(magnification 200×). Red arrow indicates apoptotic cells; blue arrow indicates viable cells. (B, C) Quantification of the effect of NaHS treatment on the
apoptosis rate in the ischemic (B) core and (C) peri-infarct regions. Data are presented as mean ± standard deviation (n = 8 rats in each group). * P < 0.001 vs.
the control group, # P < 0.001 vs. the NaHS 30 group.
NaHS) that had been suggested previously by Li et al. and Ren et al [
]. Moreover, no
study to date has assessed the relationship between the blood concentration and the
antioxidant effects of H2S. Further studies are therefore needed to more precisely evaluate the
relationship between the timing of H2S delivery and its subsequent blood concentration.
1H-MRS can sensitively detect metabolites in vivo and monitor their temporal changes
during stroke. NAA, which is predominantly present in neurons, has been proposed as a marker
of neuronal density and viability [
]. NGT is used as a predictive marker of ischemic severity
12 / 16
]. In our current results, the NAA and the NGT levels in the peri-infarct region of the
subject rats were higher in the NaHS-1 group than in the controls at 9 h post-ischemia. However,
there were no differences in these parameters in any of the animal groups at 24 h
post-ischemia. Possible reasons for this finding may be the evolution of stroke injury and the technical
limitations of the analytical methods we used. As oxidative stress and glutamate-mediated
excitotoxicity are sustained during the evolution of cytotoxic/vasogenic edema [33±35],
disruption of the blood brain barrier (BBB) and the plasma membrane ion transport function
gradually occurs. Therefore, although the protective effects of H2S can attenuate oxidative
stress and glutamate-mediated excitotoxicity, it appears that this will only delay the eventual
cell death that arises from continuous damage to different areas of the brain. Notably, we only
measured the NAA and the NGT levels in limited regions of the rat brain and more wide
ranging analysis on the neuroprotective mechanism of H2S will therefore be needed. An
investigation using multi-voxel MRS may further elucidate this phenomenon.
We have evaluated the protective effects of H2S administration prior to reperfusion and
assessed the relationship between the timing of H2S delivery and the subsequent
neuroprotective effects against I/R injury in a rat model. Neuroprotective effects of H2S against edema and
apoptosis were observed. Furthermore, our results support our hypothesis that the therapeutic
timing of H2S exposure affects its neuroprotective impact against I/R-induced cerebral injury.
Nevertheless, further studies on the precise time-dependence of H2S efficacy against cerebral
I/R injury and the mechanism underlying its neuroprotective effects are needed to assess its
potential clinical application.
S1 Table. Result values of infarct volume, peri-infarct volume, and MLS in Fig 3.
S2 Table. Result values of rADC in Fig 4.
S3 Table. Result values of rT2 in Fig 5.
S4 Table. Result values of cNAA and cNGT (ischemic core) in Fig 6.
S5 Table. Result values of cNAA and cNGT (peri-infarct region) in Fig 7.
S6 Table. Result values of apoptosis rate in Fig 8.
S1 File. NC3Rs ARRIVE guidelines checklist.
The authors gratefully acknowledge the technical support from Biomedical Imaging
Infrastructure, Department of Radiology, Asan Medical Center.
13 / 16
Conceptualization: Chul-Woong Woo, Yoonseok Choi, Dong-Cheol Woo.
Data curation: Chul-Woong Woo, Jae-Im Kwon, Woo-Hyun Shim, Yu Sub Sung.
Formal analysis: Chul-Woong Woo.
Funding acquisition: Hyun Kwon Ha, Dong-Cheol Woo.
Investigation: Chul-Woong Woo, Jae-Im Kwon, Kyung-Won Kim, Jeong-Kon Kim,
Tae Kim, Su Jeong Ham.
Methodology: Chul-Woong Woo, Jae-Im Kwon, Sang-Beom Jeon, Jinil Kim.
Project administration: Jae-Im Kwon, Yoonseok Choi.
Supervision: Kyung-Won Kim, Jeong-Kon Kim, Sang-Beom Jeon, Seung-Chae Jung,
Choong-Gon Choi, Hyun Kwon Ha, Dong-Cheol Woo.
Validation: Yoonseok Choi, Dong-Cheol Woo.
Visualization: Chul-Woong Woo.
Writing ± original draft: Chul-Woong Woo, Jae-Im Kwon.
Writing ± review & editing: Chul-Woong Woo, Kyung-Won Kim, Yoonseok Choi,
14 / 16
15 / 16
1. Ginsberg MD . Expanding the concept of neuroprotection for acute ischemic stroke: The pivotal roles of reperfusion and the collateral circulation . Progress in neurobiology. 2016 ; 145 ± 146 : 46 ± 77 . https://doi. org/10.1016/j.pneurobio. 2016 . 09 .002 PMID: 27637159 .
2. Palaniswami M , Yan B . Mechanical Thrombectomy Is Now the Gold Standard for Acute Ischemic Stroke: Implications for Routine Clinical Practice . Interventional neurology . 2015 ; 4 ( 1 ±2): 18 ± 29 . https:// doi.org/10.1159/000438774 PMID: 26600793; PubMed Central PMCID : PMC4640090 .
3. Nour M , Scalzo F , Liebeskind DS . Ischemia-reperfusion injury in stroke . Interventional neurology . 2013 ; 1 ( 3 ±4): 185 ± 99 . https://doi.org/10.1159/000353125 PMID: 25187778; PubMed Central PMCID : PMC4031777 .
4. Zhao H , Wang R , Tao Z , Gao L , Yan F , Gao Z , et al. Ischemic postconditioning relieves cerebral ischemia and reperfusion injury through activating T-LAK cell-originated protein kinase/protein kinase B pathway in rats . Stroke; a journal of cerebral circulation . 2014 ; 45 ( 8 ): 2417 ± 24 . https://doi.org/10.1161/ STROKEAHA.114.006135 PMID: 25013016 .
5. Bonaventura A , Liberale L , Vecchie A , Casula M , Carbone F , Dallegri F , et al. Update on Inflammatory Biomarkers and Treatments in Ischemic Stroke . International journal of molecular sciences . 2016 ; 17 ( 12 ). https://doi.org/10.3390/ijms17121967 PMID: 27898011; PubMed Central PMCID : PMC5187767 .
6. Gregorakos L , Dimopoulos G , Liberi S , Antipas G . Hydrogen-Sulfide PoisoningÐManagement and Complications . Angiology. 1995 ; 46 ( 12 ): 1123 ± 31 . https://doi.org/10.1177/000331979504601208 PubMed PMID: WOS:A1995TJ67300008 . PMID: 7495318
7. Yang G , Wu L , Jiang B , Yang W , Qi J , Cao K , et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase . Science . 2008 ; 322 ( 5901 ): 587 ± 90 . https://doi.org/10. 1126/science.1162667 PMID: 18948540; PubMed Central PMCID : PMC2749494 .
8. Papapetropoulos A , Pyriochou A , Altaany Z , Yang GD , Marazioti A , Zhou ZM , et al. Hydrogen sulfide is an endogenous stimulator of angiogenesis . Proceedings of the National Academy of Sciences of the United States of America . 2009 ; 106 ( 51 ): 21972 ±7. https://doi.org/10.1073/pnas.0908047106 PubMed PMID: WOS:000272994200091. PMID: 19955410
9. Kimura H. The physiological role of hydrogen sulfide and beyond . Nitric oxide: biology and chemistry / official journal of the Nitric Oxide Society . 2014 ; 41 :4± 10 . https://doi.org/10.1016/j.niox. 2014 . 01 .002 PMID: 24491257 .
10. Stein A , Bailey SM . Redox Biology of Hydrogen Sulfide: Implications for Physiology, Pathophysiology, and Pharmacology . Redox biology . 2013 ; 1(1):32±9 . https://doi.org/10.1016/j.redox. 2012 . 11 .006 PMID: 23795345; PubMed Central PMCID : PMC3685875 .
11. Yin J , Zeng QH , Shen Q , Yang XS . [ Neuroprotective mechanism of hydrogen sulfide after cerebral ischemia-reperfusion in rats] . Zhonghua yi xue za zhi . 2013 ; 93 ( 11 ): 868 ± 72 . PMID: 23859398 .
12. Yin J , Tu C , Zhao J , Ou D , Chen G , Liu Y , et al. Exogenous hydrogen sulfide protects against global cerebral ischemia/reperfusion injury via its anti-oxidative, anti-inflammatory and anti-apoptotic effects in rats . Brain research . 2013 ; 1491 : 188 ± 96 . https://doi.org/10.1016/j.brainres. 2012 . 10 .046 PMID: 23123706 .
13. Li XJ , Li CK , Wei LY , Lu N , Wang GH , Zhao HG , et al. Hydrogen sulfide intervention in focal cerebral ischemia/reperfusion injury in rats . Neural Regen Res . 2015 ; 10 ( 6 ): 932 ±7. https://doi.org/10.4103/ 1673 - 5374 .158353 PMID: 26199610; PubMed Central PMCID : PMC4498355 .
14. Gheibi S , Aboutaleb N , Khaksari M , Kalalian-Moghaddam H , Vakili A , Asadi Y , et al. Hydrogen sulfide protects the brain against ischemic reperfusion injury in a transient model of focal cerebral ischemia . Journal of molecular neuroscience: MN . 2014 ; 54 ( 2 ): 264 ± 70 . https://doi.org/10.1007/s12031-014- 0284-9 PMID: 24643521 .
15. Jang H , Oh MY , Kim YJ , Choi IY , Yang HS , Ryu WS , et al. Hydrogen sulfide treatment induces angiogenesis after cerebral ischemia . Journal of neuroscience research . 2014 ; 92 ( 11 ): 1520 ±8. https://doi. org/10.1002/jnr.23427 PMID: 24939171 .
16. Iihoshi S , Honmou O , Houkin K , Hashi K , Kocsis JD . A therapeutic window for intravenous administration of autologous bone marrow after cerebral ischemia in adult rats . Brain research . 2004 ; 1007 ( 1 ± 2): 1±9 . https://doi.org/10.1016/j.brainres. 2003 . 09 .084 PMID: 15064130 .
17. Cheng J , Alkayed NJ , Hurn PD . Deleterious effects of dihydrotestosterone on cerebral ischemic injury . Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism . 2007 ; 27 ( 9 ): 1553 ± 62 . https://doi.org/10.1038/sj.jcbfm.9600457 PMID: 17311081; PubMed Central PMCID : PMC2274912 .
18. Xing B , Chen H , Zhang M , Zhao D , Jiang R , Liu X , et al. Ischemic post-conditioning protects brain and reduces inflammation in a rat model of focal cerebral ischemia/reperfusion . J Neurochem. 2008 ; 105 ( 5 ): 1737 ± 45 . https://doi.org/10.1111/j.1471- 4159 . 2008 . 05276 . x PMID : 18248611 .
19. Prichard JW . The ischemic penumbra in stroke: prospects for analysis by nuclear magnetic resonance spectroscopy . Research publicationsÐAssociation for Research in Nervous and Mental Disease . 1993 ; 71 : 153 ± 74 . PMID: 8417465 .
20. Berthet C , Lei H , Gruetter R , Hirt L. Early predictive biomarkers for lesion after transient cerebral ischemia . Stroke; a journal of cerebral circulation . 2011 ; 42 ( 3 ): 799 ± 805 . https://doi.org/10.1161/ STROKEAHA.110.603647 PMID: 21293024 .
21. Li XJ , Li CK , Wei LY , Lu N , Wang GH , Zhao HG , et al. Hydrogen sulfide intervention in focal cerebral ischemia/reperfusion injury in rats . Neural Regen Res . 2015 ; 10 ( 6 ): 932 ±7. https://doi.org/10.4103/ 1673 - 5374 .158353 PubMed PMID: WOS: 000357298200024 . PMID: 26199610
22. Walberer M , Blaes F , Stolz E , Muller C , Schoenburg M , Tschernatsch M , et al. Midline-shift corresponds to the amount of brain edema early after hemispheric strokeÐan MRI study in rats . Journal of neurosurgical anesthesiology . 2007 ; 19 ( 2 ): 105 ± 10 . https://doi.org/10.1097/ANA.0b013e31802c7e33 PMID: 17413996 .
23. Juenemann M , Braun T , Doenges S , Nedelmann M , Mueller C , Bachmann G , et al. Aquaporin-4 autoantibodies increase vasogenic edema formation and infarct size in a rat stroke model . Bmc Immunol . 2015 ; 16 . doi: ARTN 30 https://doi.org/10.1186/s12865-015-0087-y PubMed PMID: WOS:000354835500001. PMID: 25986484
24. Barber PA , Hoyte L , Kirk D , Foniok T , Buchan A , Tuor U . Early T1- and T2-weighted MRI signatures of transient and permanent middle cerebral artery occlusion in a murine stroke model studied at 9 .4 T. Neurosci Lett . 2005 ; 388 ( 1 ): 54 ±9. https://doi.org/10.1016/j.neulet. 2005 . 06 .067 PubMed PMID: WOS: 000231743100011 . PMID: 16055267
25. Lei H , Berthet C , Hirt L , Gruetter R . Evolution of the neurochemical profile after transient focal cerebral ischemia in the mouse brain . Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism . 2009 ; 29 ( 4 ): 811 ±9. https://doi.org/10.1038/ jcbfm. 2009 .8 PMID: 19223915 .
26. Lu M , Hu LF , Hu G , Bian JS . Hydrogen sulfide protects astrocytes against H2O2-induced neural injury via enhancing glutamate uptake . Free Radical Bio Med . 2008 ; 45 ( 12 ): 1705 ± 13 . https://doi.org/10.1016/ j.freeradbiomed. 2008 . 09 .014 PubMed PMID: WOS: 000261721800011 . PMID: 18848879
27. O 'Shea JM , Williams SR , van Bruggen N , Gardner-Medwin AR . Apparent diffusion coefficient and MR relaxation during osmotic manipulation in isolated turtle cerebellum . Magnetic resonance in medicine. 2000 ; 44 ( 3 ): 427 ± 32 . PMID: 10975895 .
28. Neumann-Haefelin T , Kastrup A , de Crespigny A , Yenari MA , Ringer T , Sun GH , et al. Serial MRI after transient focal cerebral ischemia in rats: dynamics of tissue injury, blood-brain barrier damage, and edema formation . Stroke; a journal of cerebral circulation . 2000 ; 31 ( 8 ): 1965 ± 72; discussion 72±3 . PMID: 10926965 .
29. Ji Y , Pang QF , Xu G , Wang L , Wang JK , Zeng YM . Exogenous hydrogen sulfide postconditioning protects isolated rat hearts against ischemia-reperfusion injury . European journal of pharmacology . 2008 ; 587 ( 1 ±3): 1±7 . https://doi.org/10.1016/j.ejphar. 2008 . 03 .044 PMID: 18468595 .
30. Elrod JW , Calvert JW , Morrison J , Doeller JE , Kraus DW , Tao L , et al. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function . Proceedings of the National Academy of Sciences of the United States of America . 2007 ; 104 ( 39 ): 15560 ±5. https://doi.org/ 10.1073/pnas.0705891104 PMID: 17878306; PubMed Central PMCID : PMC2000503 .
31. Toombs CF , Insko MA , Wintner EA , Deckwerth TL , Usansky H , Jamil K , et al. Detection of exhaled hydrogen sulphide gas in healthy human volunteers during intravenous administration of sodium sulphide . British journal of clinical pharmacology . 2010 ; 69 ( 6 ): 626 ± 36 . Epub 2010/06/23. https://doi.org/ 10.1111/j.1365- 2125 . 2010 . 03636 . x PMID : 20565454 ; PubMed Central PMCID : PMCPmc2883755 .
32. Ren CL , Du AL , Li DL , Sui JW , Mayhan WG , Zhao HG . Dynamic change of hydrogen sulfide during global cerebral ischemia-reperfusion and its effect in rats . Brain research . 2010 ; 1345 : 197 ± 205 . https:// doi.org/10.1016/j.brainres. 2010 . 05 .017 PubMed PMID: WOS: 000280655400019 . PMID: 20478278
33. Coyle JT , Puttfarcken P . Oxidative stress, glutamate, and neurodegenerative disorders . Science . 1993 ; 262 ( 5134 ): 689 ± 95 . PMID: 7901908 .
34. Khanna A , Kahle KT , Walcott BP , Gerzanich V , Simard JM . Disruption of ion homeostasis in the neurogliovascular unit underlies the pathogenesis of ischemic cerebral edema . Translational stroke research . 2014 ; 5 ( 1 ):3± 16 . https://doi.org/10.1007/s12975-013-0307-9 PMID: 24323726; PubMed Central PMCID : PMC3946359 .
35. Oh SM , Betz AL . Interaction between Free-Radicals and Excitatory Amino-Acids in the Formation of Ischemic Brain Edema in Rats. Stroke; a journal of cerebral circulation . 1991 ; 22 ( 7 ): 915 ± 21 . PubMed PMID : WOS: A1991FW79100015 .