The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury Induced by Experimental Intracerebral Hemorrhage

R. D. Martin et al. (eds.), Subarachnoid Hemorrhage, Acta Neurochirurgica Supplement 127, 105
https://doi.org/10.1007/978-3-030-04615-6_17, © Springer Nature Switzerland AG 2020
Abstract The protein kinase RNA-like endoplasmic reticu￾lum kinase (PERK) pathway, which is a branch of the
unfolded protein response, participates in a range of patho￾physiological processes of neurological diseases. However,
few studies have investigated the role of the PERK in intrace￾rebral hemorrhage (ICH). The present study evaluated the
role of the PERK pathway during the early phase of ICH￾induced secondary brain injury (SBI) and its potential mecha￾nisms. An autologous whole blood ICH model was established
in rats, and cultured primary cortical neurons were treated
with oxyhemoglobin to mimic ICH in vitro. We found that
levels of phosphorylated alpha subunit of eukaryotic transla￾tion initiation factor 2 (p-eIF2α) and activating transcription
factor 4 (ATF4) increased significantly and peaked at 12 h
during the early phase of the ICH. To further elucidate the
role of the PERK pathway, we assessed the effects of the
PERK inhibitor, GSK2606414, and the eIF2α dephosphory￾lation antagonist, salubrinal, at 12 h after ICH both in vivo
and in  vitro. Inhibition of PERK with GSK2606414 sup￾pressed the protein levels of p-eIF2α and ATF4, resulting in
increase of transcriptional activator CCAAT/enhancer-bind￾ing protein homologous protein (CHOP) and caspase-12,
which promoted apoptosis and reduced neuronal survival.
Treatment with salubrinal yielded opposite results, which
suggested that activation of the PERK pathway could pro￾mote neuronal survival and reduce apoptosis. In conclusion,
the present study has demonstrated the neuroprotective effects
of the PERK pathway during the early phase of ICH-induced
SBI.  These findings highlight the potential value of PERK
pathway as a therapeutic target for ICH.
Keywords Intracerebral hemorrhage · Endoplasmic reticu￾lum stress · Unfolded protein response · PERK pathway ·
Neuroprotection
Introduction
Stroke, also known as a cerebrovascular accident, is a morbid
state produced by insufficient blood flow to meet the meta￾bolic demands of the brain. Intracerebral hemorrhage (ICH) is
the deadliest type of stroke with a 30-day mortality up to 40%
and severe disability in the majority of survivors [1]. The
mechanisms of ICH are extremely complex, including pri￾mary brain injury and secondary brain injury (SBI). At pres￾ent, it is generally accepted that SBI plays a more critical role
in the poor prognosis of hemorrhagic stroke. Unfortunately,
we currently have no effective solutions to SBI, which involves
oxidation, inflammation, apoptosis, and hematotoxicity [2].
SBI results in disruption of cellular metabolism and activation
of a series of stress responses such as the unfolded protein
response (UPR) in endoplasmic reticulum (ER) stress [3].
The ER is an important subcellular organelle in eukary￾otic cells. It plays a vital role in many cellular processes that
include folding of newly synthesized secretory and mem￾brane proteins, posttranslational modifications, and regula￾tion of intracellular Ca2+ homeostasis [4]. Normally, only
properly folded proteins are transported from the ER to the
Golgi apparatus; unfolded or misfolded proteins are
degraded. ER stress occurs when unfolded or misfolded
The PERK Pathway Plays a Neuroprotective Role During the Early
Phase of Secondary Brain Injury Induced by Experimental
Intracerebral Hemorrhage
Juyi Zhang#
, Peng Zhang#
, Chengjie Meng, Baoqi Dang, Haiying Li, Haitao Shen, Zhong Wang, Xiang Li, and Gang Chen
J. Zhang · P. Zhang · H. Li · H. Shen · Z. Wang (*) · X. Li (*)
G. Chen
Department of Neurosurgery and Brain and Nerve Research
Laboratory, The First Affiliated Hospital of Soochow University,
Suzhou, China
e-mail: [email protected]
C. Meng
Department of Neurosurgery and Brain and Nerve Research
Laboratory, The First Affiliated Hospital of Soochow University,
Suzhou, China
Department of Neurosurgery, Yancheng First People’s Hospital,
Yancheng, China
B. Dang
Department of Rehabilitation Medicine, Zhangjiagang Hospital of
Traditional Chinese Medicine, Suzhou, China
These authors contributed equally to this work.
proteins accumulate and the folding capacity of ER chaper￾ones exceeds the capacity of the ER lumen to facilitate their
disposal. As a consequence, a battery of adaptive processes,
collectively known as the UPR, can be activated that transmit
signals from the ER to the cytosol and nucleus to combat
harmful effects of ER stress and restore normal cellular
homeostasis [5]. The UPR can remove unfolded or misfolded
proteins when ER stress occurs, and it might play a signifi￾cant role in cell survival [6]. However, if stimuli are severe or
prolonged, ER stress responses may be unable to compen￾sate, and cell apoptosis may be induced [7].
The UPR is triggered by activation of three sensor pro￾teins at the ER membrane: activating transcription factor-6
(ATF6), inositol-requiring enzyme-1 (IRE1), and protein
kinase RNA-like ER kinase (PERK) [8]. Activated PERK
phosphorylates the alpha subunit of eukaryotic translation
initiation factor 2 (eIF2α), which can block the initiation
stage of translation, thereby reducing protein synthesis and
decreasing the ER load [9]. If ER stress is sustained, the
ER-specific apoptosis pathway is activated by promoting
expression of transcriptional activator CCAAT/enhancer￾binding protein homologous protein (CHOP) and cas￾pase-12 (CASP12) [10]. In recent years, several studies
have reported that the UPR plays a vital role in the fate of
neuronal cells following ischemic stroke. Although ICH
only accounts for 10–20% of all cerebrovascular accidents
worldwide [11], it is the most devastating type of stroke
with a high morbidity and mortality; up to 50% of patients
die within the first 24 h [12].
It is not clear whether ER stress and the UPR are involved
in mechanisms that underlie ICH-induced SBI. The purpose
of this study was to investigate the role of the PERK pathway
during the early phase of ICH-induced SBI and its potential
mechanisms. We monitored the time course of expression of
the PERK pathway and utilized two experimental tools,
PERK inhibitor GSK2606414 [13, 14] and eIF2α dephos￾phorylation inhibitor salubrinal [15, 16], which exert oppo￾site effects both in vivo and in vitro.
Materials and Methods
Animals
Adult male Sprague-Dawley rats (250–300 g, Animal Center
of the Chinese Academy of Sciences, Shanghai, China) were
raised with free access to water and food and housed in tem￾perature- and humidity-controlled animal quarters with a 12-h
light/dark cycle. All animal experiments were approved by the
Ethics Committee of the First Affiliated Hospital of Soochow
University and in accordance with the National Institutes of
Health Guide.
ICH Model
The ICH model was established in rats using stereotaxic
injection of autologous whole blood according to a previous
report [17] with some modifications. In brief, rats were anes￾thetized and then mounted on a stereotaxic frame
(ZH-Lanxing B type, Anhui Zhenghua Biological Equipment
Co. Ltd. Anhui, China). Then, a cranial burr hole was drilled
0.2 mm anterior to bregma and 3.5 mm lateral to the midline,
which corresponded to the right basal ganglia. Autologous
whole blood (100 μL) was collected by cardiac puncture and
injected slowly (5.5  mm ventral to the cortical surface,
20 μL/min) with a microinjector (Hamilton Company, NV,
USA). To prevent reflux, the needle was kept in place for an
additional 5 min. The bone hole was sealed with bone wax,
and the scalp was then disinfected and sutured. During the
entire surgery, rats were placed on a heating pad in a supine
position, and the pad was maintained at ~27–35  °C.  Vital
signs were monitored continuously. After establishment of
the ICH model, the rats were returned to their cages with
food and water. A representative brain coronal section was
shown in Fig. 1a.
Experimental Design
There were two types of in vivo experiments. In experiment
1, we analyzed the time course of changes in levels of
p-eIF2α and ATF4 after ICH. A total of 72 rats (80 rats were
used, 72 rats survived after surgery) were randomly (used the
randomization table) divided into six groups of 12 rats per
group, which included a sham group and five experimental
groups arranged by time after ICH: 4, 8, 12, 16, and 24 h. At
the indicated time point after ICH, rats were killed, and the
brain samples of six rats in each group were dissected and
used for Western blot analysis. Double immunofluorescence
staining of p-eIF2α and ATF4 with neuronal nuclei (NeuN)
was performed in the sham group and 12  h after ICH
(Fig. 1b).
In experiment 2, 108 rats (129 rats were used, 108 rats
survived) were randomly (used the randomization table)
divided into six groups of 18 rats per group: sham, ICH,
ICH  +  vehicle (for GSK2606414), ICH  +  GSK2606414
(90 μg in 5 μL sterile saline), ICH + vehicle (for salubrinal),
and ICH + salubrinal (1 mg/kg body weight). Neurological
scoring and brain edema were assessed at 12  h after
ICH.  Expression levels of p-eIF2α, ATF4, CHOP, and
CASP12 were determined by Western blot analysis at 12 h
after ICH.  Finally, terminal deoxynucleotidyl transferase￾mediated dUTP nick end labeling (TUNEL) and fluoro-jade
B (FJB) staining were also performed at 12 h after ICH in
each group (Fig. 1c).
J. Zhang et al.
107
In experiment 3, primary rat cortical neurons were treated
with oxyhemoglobin (OxyHb) (10 μmol/L) to mimic effects
of ICH in  vitro. The experimental groups were similar to
those of experiment 2 in vivo, and we assessed changes in
protein levels of p-eIF2α, ATF4, CHOP, and cleaved CASP12.
At 12 h after OxyHb treatment, a sulforhodamine B (SRB)
assay was used to test cell viability, and the cell culture super￾natants were collected for lactate dehydrogenase (LDH)
activity detection. Double immunofluorescence staining of
TUNEL and NeuN was performed in all groups (Fig. 1d).
For neurological scoring and brain edema evaluation, the
observers did not know group of rats, either the component of
infusion. For Western blot analysis, the bands were collected
from one independent experiment using one rat, and the sta￾tistical data were from at least six rats. For all the immuno￾fluorescence analysis, the representative images were from at
least three independent experiments using six rats.
Antibody Characterization and Drugs
Anti-p-eIF2α antibody (ab32157), anti-eIF2α antibody
(ab169528), anti-CHOP antibody (ab11419), anti-CASP12
antibody (ab62484), mouse anti-NeuN monoclonal antibody
(ab104224), and anti-β-tubulin antibody (ab179513) were
purchased from Abcam (Cambridge, MA, USA). Anti-ATF4
antibody (sc-200) was purchased from Santa Cruz (Santa
Cruz, CA, USA). Salubrinal and GSK2606414 were pur￾chased from TargetMol (Boston, MA, USA).
Drug Administration
One hour after surgery, the PERK pathway inhibitor,
GSK2606414, was dissolved in dimethyl sulfoxide (DMSO)
Immunofluorescence
Time course analysis of the expression of
PERK pathway after ICH
8h 12h 16h 24h
- Vehicle
(GSK2606414) Vehicle
(GSK2606414)
Vehicle
(salubrinal) Vehicle
(salubrinal)
salubrinal
salubrinal
n=18
n=12 n=12 n=12 n=12 n=12 n=12
n=18 n=18
ATF4
CHOP CHOP
Neurological scoring
Brain edema
Effects of PERK pathway on ICH-induced SBI
and the potential mechanisms
Effects of PERK pathway on neurons subjected
to OxyHb and the potential mechanisms
cleaved-CASP12 cleaved-CASP12
n=18 n=18
Fig. 1 Intracerebral hemorrhage model and experimental design. (a)
Representative whole brains and brain slices from ICH model rats. (b)
Experiment 1 was designed to evaluate expression of p-eIF2α and
ATF4 at different time points. (c) Experiment 2 was designed to inves￾tigate effects of the PERK pathway on ICH-induced SBI and potential
mechanisms. (d) Experiment 3 was designed to investigate the role of
the PERK pathway in vitro
The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury…
108
and further diluted in sterile saline to a final concentration of
0.5%. Five microliters of GSK2606414 (90  μg) was then
administered intracerebroventricularly at a rate of 0.5  μL/
min [18]. The microsyringe was left in situ for another
10  min before being removed slowly. The eIF2α dephos￾phorylation inhibitor, salubrinal, was infused intraperitone￾ally at a dose of 1 mg/kg in saline with 1.5% DMSO [19].
Equal volumes of DMSO solutions were respectively admin￾istered to vehicle control animals.
Intracerebroventricular Injection
Intracerebroventricular injection was conducted as reported
previously [20]. Briefly, rats were placed in a stereotaxic
frame after anesthetization as described above. Then, a small
burr hole was drilled into the skull 1.0  mm lateral to and
1.5 mm posterior to bregma over the left hemisphere. The
needle of a 10  μL Hamilton syringe was slowly inserted
through the burr hole into the left lateral ventricle 4.0 mm
below the dural surface. A reagent was infused into the left
lateral ventricle at a rate of 0.5 μL/min.
Establishment of the In Vitro ICH Model
and Cell Treatment
Isolation and culture of primary cortical neurons has
been described previously [21, 22]. Briefly, whole brains
of 17-day rat embryos were used to prepare primary
neuron-enriched cultures. Every effort was made to mini￾mize the number of embryos used and their suffering.
First, we removed the blood vessels and the meninges.
Then, the brain tissues were digested with 0.25% trypsin
for 5  min at 37  °C.  After termination of digestion, the
suspension was centrifuged at 1500 rpm for 5 min, and
the pellet was resuspended in plates and cultured in
Neurobasal Medium (GIBCO, Carlsbad, CA, USA).
Cultures were maintained in an atmospheric incubator at
37 °C with 5% CO2. Neurons were cultured for 2 weeks,
and half of the media was replaced every 2  days. To
mimic ICH, neurons were treated with 10  μM OxyHb
[23]. The cultures were divided into four groups as fol￾lows: control; OxyHb treatment for 12 h; OxyHb + vehi￾cle (for GSK2606414), pretreatment with GSK2606414
(1 μM) for 1 h, thorough rinsing, and OxyHb treatment
for 12 h [24]; OxyHb + vehicle (for salubrinal); and pre￾treatment with salubrinal (50 μM) for 1 h, thorough rins￾ing, and OxyHb treatment for 12 h [25].
Neurological Scoring
At 12 h after ICH, rats in experiment 2 were assessed by neu￾rological scoring before euthanasia. All rats were evaluated
using a previously published scoring system that monitored
appetite, activity, and neurological deficits [21] (Table 1).
Brain Edema
The index of brain edema was determined using the wet/dry
method as described previously [26]. Briefly, the brain tissue
was removed and collected, and the samples were weighed
immediately (wet weight), followed by drying at 100 °C for
72 h. And then the tissues were reweighted to obtain the dry
weight. The percentage of water content was calculated as
follows: [(wet weight − dry weight)/wet weight] × 100%.
Cell Viability
Neuronal viability was evaluated by SRB assay. Following
treatment incubation, the culture medium was removed, and
neurons were fixed with 10% trichloroacetic acid (TCA) fol￾lowed by staining with 0.4% SRB. Absorbances were mea￾sured at 540  nm with a Bio-Rad Microplate reader. Cell
viability was measured in triplicate and repeated at least
three independent times.
LDH Assay
The concentrations of LDH in the culture medium were mea￾sured using a LDH detecting kit (A020-2; Jiancheng Biotech,
Nanjing, China) according to the instructions. The data were
presented relative to standard curves.
Table 1 Behavior and activity scores
Category Behavior Score
Appetite Finished meal
Left meal unfinished
Scarcely ate
Activity Walk and reach at least three corners of the cage
Walk with some stimulations
Almost always lying down
Deficits No deficits
Unstable walk
Impossible to walk
J. Zhang et al.
Western Blot Analysis
After perihematomal tissues were collected, the brain samples
of each animal were homogenized separately and then
mechanically lysed in lysis buffer (Beyotime Institute of
Biotechnology, Jiangsu, China). After centrifuging at
15000 × g for 10 min at 4 °C, the supernatants were collected
immediately. Protein concentration was determined using an
enhanced bicinchoninic acid (BCA) protein assay kit
(Beyotime Institute of Biotechnology). Then, the protein
(30 μg/lane) were loaded on a 10% SDS-PAGE gel, separated,
and then electrophoretically transferred to a polyvinylidene
difluoride (PVDF) membrane (Millipore Corporation,
Billerica, MA, USA). The membrane was blocked with 5%
bovine serum albumin (Biosharp, Hefei, AH, China) for 1 h at
room temperature and then probed with the primary antibody
overnight at 4 °C. Next, the membrane was incubated with the
corresponding HRP-conjugated secondary antibody for 2 h at
37 °C and then washed with phosphate buffer saline (PBS)-
Tween20 (PBST). Finally, bands were visualized by enhanced
chemiluminescence (ECL) as reported previously [26] and
analyzed using ImageJ software. Relative quantity of proteins
was determined by normalizing to levels of loading controls.
Immunofluorescence Microscopy
Brain tissues were fixed in 4% paraformaldehyde and embed￾ded in paraffin. The tissues were cut into 4 μm sections and
dewaxed immediately before immunofluorescence staining.
Double immunofluorescence was performed with primary
antibodies for p-eIF2α or ATF4 and NeuN.  After washing
three times with PBS, the samples were stained with appro￾priate secondary antibodies. All primary antibodies were
applied at a dilution of 1:100, and all secondary antibodies
were diluted 1:500. Normal rabbit IgG was used as a nega￾tive control (data not shown). Sections were observed with a
fluorescence microscope (BX50/BX-FLA/DP70, Olympus
Co., Japan), and relative fluorescence intensity was analyzed
as described previously [27].
TUNEL Staining
Quantitation of apoptotic cells was performed using TUNEL
staining according to the manufacturer’s protocol (DeadEnd
Fluorometric Kit, Promega, WI, USA). Three sections per rat
were examined and photographed in parallel for TUNEL￾positive cell counting.
FJB Staining
FJB staining was used to reveal the neuronal degradation,
which was sensitive and highly specific [28]. The procedures
were performed as previously described [29]. In brief, the
brain sections were deparaffinized and then dried in an oven.
Then, sections were rehydrated using xylene and a series of
graded ethanol solutions followed by water. Brain sections
were permeabilized in 0.04% Triton X-100 and incubated
with FJB dye solution. Then they were observed and photo￾graphed in parallel by a fluorescence microscope (BX50/
BX-FLA/DP70, Olympus Co.). The FJB-positive cell num￾bers were counted after being observed and photographed in
parallel for six microscopic fields in each tissue. Microscopy
was performed by an observer blind to the experimental
group.
Statistical Analysis
GraphPad Prism 7 was used for all statistical analy￾sis. Neurobehavioral scoring is presented as the median
with the interquartile range. All other data represent
mean ± SEM. One-way ANOVA for multiple comparisons
and the Student-Newman-Keuls post hoc test were used to
assess differences among all groups. Differences were con￾sidered significant at p < 0.05.
Post hoc power analysis was performed according to a
power analysis (PRISM, t-test comparison of the mean).
Based on a two-sample t-test with a specified mean difference
between the sham and ICH group, an estimated standard
deviation was calculated, and alpha = 0.05, power > 0.75 for
a sample size of n = 6 per groups. We assigned six rats in each
groups because this number was close to the prediction.
Results
Elevation of p-eIF2α and ATF4 Levels
in Brain Tissues After ICH
In experiment 1, the Western blot analysis showed that the
ICH group expressed higher protein levels of p-eIF2α and
ATF4 compared with the sham group. After induction of
ICH, protein levels of p-eIF2α and ATF4  in brain tissues
were significantly elevated at 4 h and peaked at 12 h, which
were remarkably higher in the 12 h group compared with the
8  h and 16  h groups (Fig.  2a, b). Double immunofluores￾The PERK Pathway Plays
Fig. 2 Protein levels of p-eIF2α and ATF4 in brain tissues after ICH.
(a) Western blot analysis and quantification of p-eIF2α and eIF2α pro￾tein levels at different time points following ICH in brain tissues. (b)
Western blot analysis and quantification of ATF4 protein levels at dif￾ferent time points following ICH in vivo. (c) Immunofluorescence in
brain tissues. Double immunofluorescence was performed with p-eIF2α
antibodies (green) and a neuronal marker (NeuN, red). Nuclei were
fluorescently labeled with DAPI (blue). Scale bar  =  30  μm. (d)
Immunofluorescence in brain tissues. Double immunofluorescence was
performed with ATF4 antibodies (green) and a neuronal marker (NeuN,
red). Nuclei were fluorescently labeled with DAPI (blue). Scale
bar = 30 μm. In A and B, mean values for the sham group or control
group were normalized to 1.0. One-way ANOVA followed by Student￾Newman-Keuls post hoc tests were used. Data are mean  ±  SEM. *
p < 0.05, **p < 0.01 vs. sham group; ##p < 0.01 12 h group vs. 8 h group; &p < 0.05 12 h group vs. 16 h group, n = 12
J. Zhang et al.
111
cence staining in sham and ICH groups further verified that
p-eIF2α and ATF4 were markedly expressed in neurons and
increased at 12 h after ICH (Fig. 2c, d). Hence, we focused
on the PERK pathway in neurons at 12 h after ICH in the
following studies.
PERK Pathway Activation Ameliorated
Neurological Behavior Impairment
and Brain Edema in the Early Phase of ICH
The PERK inhibitor, GSK2606414, was injected intracere￾broventricularly at 1 h after ICH, and the eIF2α dephosphor￾ylation inhibitor, salubrinal, was injected intraperitoneally at
30 min before ICH. Then the protein levels of p-eIF2α and
ATF4 were detected by Western blot. It was shown that
administering GSK2606414 significantly suppressed the
increases in protein levels of p-eIF2α and ATF4 after ICH. On
the contrary, the inhibitor of eIF-2α dephosphorylation, salu￾brinal, could significantly increase protein levels of p-eIF2α
and ATF4 (Fig. 3a–c). To assess the effects of manipulating
the PERK pathway on neurological behavioral impairment
after ICH, all rats were subjected to behavioral testing before
being killed. Remarkable neurological behavioral impair￾ment was observed in the ICH, ICH  +  vehicle (for
GSK2606414), and ICH  +  vehicle (for salubrinal) groups
compared with the sham group at 12 h after ICH. After intra￾cerebroventricular GSK2606414 injection, neurological
behavioral impairment was exacerbated. In contrast, after
intraperitoneal injection of salubrinal, neurobehavioral defi￾cits were significantly ameliorated (Fig.  3d). Furthermore,
we evaluated brain water content in each group. In the ICH
group, brain water content was significantly increased when
compared with the sham group. Salubrinal treatment signifi￾cantly reduced ICH-induced brain water content, whereas
GSK2606414 treatment significantly increased brain edema
(Fig. 3e).
PERK Pathway Activation Inhibited
Neuronal Apoptosis and Necrosis Induced by
ICH at 12 h In Vivo
It has been reported that PERK signaling pathway was
involved in ER stress-induced apoptosis. The ER-specific
apoptosis pathway is activated by promoting expression of
CHOP and CASP12 [10]. Western blot was used to measure
the protein levels of CHOP and the cleavage of CASP12.
The protein levels of CHOP and cleaved CASP12 signifi￾cantly increased at 12  h in the ICH, ICH  +  vehicle (for
GSK2606414), and ICH  +  vehicle (for salubrinal) groups
(Fig. 4a). With the administering of GSK2606414, the levels
of CHOP and cleaved CASP12 were significantly increased.
Otherwise, with the treatment of salubrinal, it showed an
opposite effect, exhibiting that significantly suppressed the
increase of CHOP and cleaved CASP12 induced by ICH
(Fig. 4a). In addition, histological examination showed that
the number of TUNEL-positive neurons and FJB-positive
cells significantly increased at 12 h in the ICH, ICH + vehi￾cle (for GSK2606414), and ICH + vehicle (for salubrinal)
groups (Fig.  4b, c). Treatment with GSK2606414 signifi￾cantly increased the total number of TUNEL and NeuN
double-stained cells at 12 h after ICH, as well as the FJB￾positive cells, compared with the ICH  +  vehicle (for
GSK2606414) group at 12  h after ICH (Fig.  4b, c).
Compared with the inhibition experiments, activation of the
PERK pathway yielded opposite results. Treatment with
salubrinal significantly lowered the total number of TUNEL
and NeuN double-stained cells at 12 h after ICH (Fig. 4b).
Similarly, the ICH + salubrinal group showed a significant
reduction in the number of FJB-positive cells compared
with the ICH + vehicle (for salubrinal) group at 12 h after
ICH (Fig. 4c).
PERK Pathway Activation Promoted
Neuronal Survival at 12 h After ICH In Vitro
In vitro, primary cortical neurons were subjected to OxyHb
to mimic the ICH model. Similar trends were observed in
expression of p-eIF2α and ATF4 with the in vivo experi￾ment after the treatment of GSK2606414 and salubrinal.
The results demonstrated that GSK2606414 treatment
could significantly suppress the increases in protein levels
of p-eIF2α and ATF4 induced by the OxyHb treatment. On
the other hand, salubrinal significantly increased protein
levels of p-eIF2α and ATF4 (Fig. 5a). Compared with the
control group, a significant decrease in neuronal viability
was observed in the OxyHb group, and this was exacer￾bated by inhibition of the PERK pathway (Fig. 5b). On the
contrary, after the treatment of salubrinal, the cell viability
was rescued in neurons under OxyHb stimulus (Fig. 5b).
Similar to the results of cell viability, the LDH release was
elevated after OxyHb stimulus. And with the treatment of
GSK2606414 and salubrinal, the release of LDH was exac￾erbated rescued respectively compared to vehicle group
(Fig. 5c).
The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury…
Fig. 3 Effects of PERK pathway on brain injury in vivo at 12 h after
ICH. (a–c) Western blot analysis showing phosphorylation levels of
eIF2α and expression of ATF4 in the sham, ICH, ICH + vehicle (for
GSK2606414), ICH  +  GSK2606414, ICH  +  vehicle (for salubrinal),
and ICH + salubrinal groups at 12 h after ICH onset. *
p < 0.05 vs. sham
group, **p < 0.01 vs. sham group; #
p < 0.05, &p < 0.05, &&p < 0.01,
n = 12. (d) Neurological scoring. ***p < 0.001 vs. sham group; #
p < 0.05, &p < 0.05, n = 18. (e) Brain water content at 12 h post-ICH. ***p < 0.001
vs. sham group; #
p < 0.05, &&p < 0.01, n = 6
J. Zhang et al.
salubrinal −−−−−+ FJB numbers/ mm2
Fig. 4 PERK pathway was involved in ICH-induced neuronal apopto￾sis and necrosis in  vivo at 12  h. (a) Western blot analysis showing
expression of CHOP and cleaved CASP12  in the sham, ICH,
ICH + vehicle (for GSK2606414), ICH + GSK2606414, ICH + vehicle
(for salubrinal), and ICH + salubrinal groups at 12 h after ICH onset.
**p < 0.01 vs. sham group; ##p < 0.01, &&p < 0.01, n = 6. (b) TUNEL
staining showing apoptotic cells in the sham, ICH, ICH + vehicle (for
GSK2606414), ICH  +  GSK2606414, ICH  +  vehicle (for salubrinal),
and ICH + salubrinal groups at 12 h after ICH onset. Double immuno￾fluorescence was performed with TUNEL (green) and a neuronal
marker (NeuN, red), and nuclei were fluorescently labeled with DAPI
(blue). Scale bar = 30 μm. The percentage of TUNEL-positive neurons
in each group. **p < 0.01 vs. sham group; #
p < 0.05, &p < 0.05, n = 12.
(c) FJB staining (green) shows neuronal degradation in the cerebral cor￾tex. Scale bar = 26 μm. Arrows indicate FJB-positive cells. FJB-positive
cells/mm2
was determined in the brain cortex at 12 h. *
p < 0.05 vs. sham
group; #
p < 0.05, &p < 0.05, n = 12
The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury…
Fig. 5 Effects of PERK-eIF2-ATF4 pathway on in OxyHb-induced
neuronal damage. (a) Western blot analysis showing phosphorylation
levels of eIF2α and expression of ATF4 in the control, OxyHb, OxyHb
+ vehicle (for GSK2606414), OxyHb + GSK2606414, OxyHb + vehi￾cle (for salubrinal), and OxyHb + salubrinal groups at 12 h. **p < 0.01
vs. control group; #
p < 0.05, &p < 0.05, &&p < 0.01, n = 6. (b) Cell viabil￾ity in neurons was measured by SRB assay. ***p  <  0.001 vs. control
group; ###p < 0.001; &&p < 0.01, n = 6. (c) LDH analysis. ***p < 0.001 vs.
control group; ###p < 0.001; &&&p < 0.001, n = 6
J. Zhang et al.
115
PERK Pathway Activation Reduced Neuronal
Apoptosis at 12 h After ICH In Vitro
Experiments performed in vitro yielded similar results. With
the treatment of GSK2606414, the protein levels of CHOP
and cleaved CASP12 were significantly increased at 12 h in
neurons after treatment with OxyHb (Fig.  6a), when
compared with the OxyHb + vehicle (for GSK2606414)
group. In addition, neurons subjected to OxyHb +
GSK2606414 showed a significant increase in neuronal
apoptosis compared with the OxyHb + vehicle (for
GSK2606414) group measured by TUNEL staining (Fig. 6b).
However, treatment with salubrinal could significantly sup￾press the expression of CHOP and cleaved CASP12 at 12 h
in neurons after treatment with OxyHb (Fig. 6a) when com￾pared with the OxyHb + vehicle (for salubrinal) group. Also,
neurons subjected to OxyHb + salubrinal showed significant
inhibition of neuronal apoptosis compared with the OxyHb +
vehicle (for salubrinal) group (Fig. 6b).
Discussion
ER stress-induced cell death is one of the most significant
causes of brain injury [30]. When ER stress occurs, cells
restore ER function by initiating a series of adaptive pro￾cesses through the UPR [31]. Previous reports have sug￾gested that the PERK pathway, which is part of the UPR,
may participate in a range of pathophysiological processes of
neurological diseases [32, 33]. However, it is unclear whether
the PERK pathway is involved in the occurrence and devel￾opment of post-ICH brain injury. Here, for the first time, we
explored a possible role of the PERK pathway during the
early phase of ICH-induced SBI both in vivo and in vitro. As
previously reported, when unfolded or misfolded protein
accumulates, PERK is activated by oligomerization and
trans-autophosphorylation [34]. Phosphorylated PERK spe￾cifically induces phosphorylation of eIF2α at ser51 (p-eIF2α),
which then upregulates transcription factor ATF4 (Fig.  7)
[35]. In experiment 1, we first investigated spatial-temporal
expression of p-elF2α and ATF4 protein after ICH. As shown
in Fig. 2a, b, under ICH condition, p-eIF2α and downstream
ATF4 showed the same trend, such that the ratio of p-elF2α/
elF2α and protein levels of ATF4 were remarkably elevated
at 4 h and peaked at 12 h. Furthermore, to investigate spatial
expression of the PERK pathway in brain tissue, as shown in
Fig.  2c, d, double immunofluorescence staining indicated
that p-eIF2α and ATF4 were markedly expressed in neurons
after ICH. This suggests that the PERK pathway was acti￾vated and that this pathway may play a vital role in ICH￾induced SBI.  Based on these findings, subsequent
experiments focused on the PERK pathway in neurons at
12 h after ICH.
It is well known that levels of phosphorylated protein can
be regulated by inhibiting both phosphorylation and dephos￾phorylation. Consistent with previous studies [36, 37], as
shown in Fig.  3a, the potent p-eIF2α dephosphorylation
inhibitor, salubrinal, significantly increased expression of
p-eIF2α and ATF4 at 12 h after ICH, whereas levels of both
proteins were decreased by the selective PERK inhibitor,
GSK2606414. Previous research has demonstrated that
p-eIF2α suppresses initiating translation of global protein
synthesis, which promotes cell survival by preventing further
accumulation of unfolded or misfolded proteins in the ER
[38, 39]. In addition, it is important for recovery from vari￾ous stresses that ATF4 triggers expression of genes involved
in amino acid metabolism, antioxidant stress, protein fold￾ing, and autophagy [40]. To define the effects of the PERK
pathway on ICH-induced neurological behavioral impair￾ment and brain edema, we performed neurological scoring
and measured brain water content.
As shown in Fig. 3d, e, rats showed severe neurological
behavioral impairment and brain edema compared with the
sham group at 12 h after ICH induction. ICH-induced neu￾rological deficits and edema were ameliorated after activat￾ing the PERK pathway with salubrinal and aggravated by
blocking the PERK pathway with GSK2606414. In addi￾tion, TUNEL and FJB staining were utilized to explore
effects of the PERK pathway on apoptosis in brain tissues at
12 h post-ICH induction. As shown in Fig. 4b, c, the num￾bers of TUNEL-positive cells and FJB-positive cells in
brain tissue around the hematomas were significantly
increased in the ICH group compared with the sham group,
and these cell numbers were augmented in the
ICH + GSK2606414 group and reduced in the ICH + salu￾brinal group. These data clearly suggest that increasing
expression of p-eIF2α and ATF4 promotes neuronal survival
and suppresses apoptosis, and this process can be reversed
by reducing these protein levels after ICH.
At least three branches participate in ER stress-induced
apoptotic events. These include the CHOP pathway [41], the
ER-associated CASP12 pathway [42], and the cJUN NH2-
terminal kinase (JNK) pathway [43, 44]. Interestingly, as
shown in Fig. 4a, GSK2606414 administration significantly
increased CHOP expression at 12 h after ICH, while treat￾ment with salubrinal remarkably suppressed expression of
The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury…
Fig. 6 PERK-eIF2-ATF4 pathway participated in OxyHb-induced neuronal apoptosis in vitro.
(a) Western blot analysis showing expression of CHOP and CASP12  in the control, OxyHb,
OxyHb + vehicle (for GSK2606414), OxyHb + GSK2606414, OxyHb + vehicle (for salubrinal),
and OxyHb + salubrinal groups at 12 h. **p < 0.01 vs. control group; ##p < 0.01; &&p < 0.01, n = 6.
(b) TUNEL staining to elucidate the role of PERK in OxyHb-treated neurons in vitro at 12 h.
Representative images from control, OxyHb, OxyHb + vehicle (for GSK2606414), OxyHb +
GSK2606414, OxyHb + vehicle (for salubrinal), and OxyHb + salubrinal groups. Each group was
subjected to OxyHb except for the control group. Scale bar = 20 μm. The percentage of TUNEL￾positive cells. **p < 0.01 vs. control group; #p < 0.05, &p < 0.05, n = 6
J. Zhang et al.
117
these pro-apoptotic proteins. CHOP, also known as growth
arrest- and DNA damage-inducible gene 153 (GADD153), is
an ER stress-specific transcription factor that consist of an
N-terminal transcriptional activation domain and a
C-terminal basic-leucine zipper [45]. Normally, CHOP is
expressed at extremely low levels. However, once ER stress
occurs, its expression significantly increases. CHOP can be
activated via the PERK-eIF2α phosphorylation pathway,
which triggers an increase in expression of ATF4. ATF4 then
binds to the site of an amino acid reaction element of the
CHOP promoter. In this study, we found that although the
PERK-eIF2α-ATF4 axis is indispensable for CHOP expres￾sion, after treatment, there is an inverse relationship between
protein levels of CHOP and expression of p-eIF2α and ATF4.
This suggests that the PERK pathway primarily plays a neu￾roprotective rather than pro-apoptotic role at 12 h after ICH.
Another important pro-apoptosis protein is CASP12,
which can induce apoptosis alone through ER stress rather
than other apoptotic pathways. Under normal physiological
conditions, CASP12 exists as an inactive zymogen similar to
other cysteine proteases. Abnormal calcium can trigger spe￾cific activation of CASP12  in the ER, which coordinates
with other ER stress molecules to activate CASP9 that trans￾mits information to CASP3, causing cells to eventually
undergo apoptosis [46]. In CASP12-deficient cells, apopto￾sis can be evoked by certain stimuli other than ER stress,
which suggests that CASP12 is a specific apoptosis factor
associated with ER stress [47]. Therefore, we selected CHOP
and CASP12 as markers of ER stress and apoptosis. As
shown in Fig. 3b, results support anti-apoptotic effects of the
PERK pathway during the early phase of ICH-induced
SBI. However, although CASP12 has been recognized as a
marker of ER stress-induced apoptosis in rats and mice,
humans lack a functional CASP12 homologue due to multi￾ple stop codons [45]. This represents a major impediment in
translation from basic experiments to clinical practice.
In many previous in  vitro studies, ER stress could be
induced by tunicamycin, which is a glycosylation inhibitor,
or thapsigargin, which is a highly selective inhibitor of the
ER Ca2+-dependent ATPase [48, 49]. To define further the
role of the PERK pathway, we established an in vitro model
of ICH by treating primary cortical neurons with OxyHb. As
shown in Figs. 5 and 6, neurons subjected to OxyHb yielded
similar results regarding TUNEL staining and expression of
p-eIF2α, ATF4, CHOP, and CASP12. Compared with the
control group, a significant decrease in neuronal viability
was observed in the OxyHb group, and viability was reduced
by GSK2606414 treatment and enhanced by salubrinal
administration. Consistent with these findings, as shown in
Fig. 6b, the necrosis index showed a trend similar to that of
the apoptotic index. Taken together, these data further con￾firm that the PERK pathway plays a neuroprotective role
during the early phase of SBI induced by ICH. In addition,
studies of ICH have increasingly recognized the significance
of particular blood components in brain injury [50]. Thus,
different responses may be induced by different blood com￾ponents. Although that OxyHb mimics ICH induction has
been well-accepted, which specific blood component is pre￾dominantly responsible for activation of the PERK pathway
is unknown.
The current study has several limitations. The generally
accepted view is that in a physiological state, the three trans￾membrane protein receptors, ATF6, IRE1, and PERK, are
bound by glucose-regulated protein 78 (GRP78), which dis￾sociates from these receptors and allows their activation
under stress conditions [51]. This point of view has been
challenged because it has been reported that unfolded or mis￾folded proteins can bind directly to ER stress sensor proteins
to activate the UPR [52]. In this study, we only focused on
effects of the PERK pathway, and initiation of the PERK
pathway after ICH requires further study. In addition, current
knowledge indicates that the UPR protects cells from ER
Fig. 7 Schematic representation of potential mechanisms of the PERK
pathway in neuroprotection under ICH conditions. Following ICH, the
activation of the ER stress response induces neuronal apoptosis.
Consequently, activated PERK pathway increases the protein levels of
p-eIF2α and ATF4. At the early phase of ICH-induced brain injury, the
PERK pathway is triggered to block the initiation process of transla￾tion, thereby reducing protein synthesis and decreasing the ER load,
which might play a significant role in neuroprotection
The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury…
118
stress by reducing synthesis of new proteins and enhancing
degradation of unfolded or misfolded proteins. However,
failure of the UPR due to severe or prolonged ER stress
eventually promotes apoptotic cell death, which is an effec￾tive measure of protecting an organism from rogue cells
expressing dysfunctional signal molecules [53].
Unfortunately, it has not been clear how the UPR globally
coordinates cytoprotective and pro-apoptotic outcomes
between a survival or death fate [54].
Based on this, we have proposed a hypothesis that the
PERK pathway predominantly plays a neuroprotective role
in the early phase and a pro-apoptotic role in the late phase
of ICH-induced SBI. In our recent study, it has been reported
that PERK pathway activation promoted ICH-induced SBI
by inducing neuronal apoptosis in the late phase [55]. And in
this study, the neuroprotective role of the PERK pathway in
the early phase has been confirmed.
Acknowledgments This work was supported by the Project of Jiangsu
Provincial Medical Innovation Team (No. CXTDA2017003), Jiangsu
Provincial Medical Youth Talent (No. QNRC2016728), Suzhou Key
Medical Centre (No. Szzx201501), Scientific Department of Jiangsu
Province (No. BE2017656), Suzhou Government (No. SYS201608 and
LCZX201601), Jiangsu Province (No. 16KJB320008), and
Zhangjiagang Science and Technology Pillar Program (ZKS1712).
Conflict of Interest: We declare that we have no conflicts of interest.
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The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury…