J Neuro Res. 2018;96:1887–1899. wileyonlinelibrary.com/journal/jnr © 2018 Wiley Periodicals, Inc | 1887
Received: 16 May 2018
|
Revised: 26 August 2018|
Accepted: 27 August 2018 DOI: 10.1002/jnr.24326R E S E A R C H A R T I C L E
Neuroprotective effects of SMTP‐44D in mice stroke model in relation to neurovascular unit and trophic coupling
Xiaowen Shi
1| Yasuyuki Ohta
1| Jingwei Shang
1| Ryuta Morihara
1| Yumiko Nakano
1| Yusuke Fukui
1| Xia Liu
1| Tian Feng
1| Yong Huang
1| Kota Sato
1| Mami Takemoto
1| Nozomi Hishikawa
1| Toru Yamashita
1| Eriko Suzuki
2| Keiji Hasumi
2| Koji Abe
11Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
2Department of Applied Biological Science, Tokyo Noko University, Fuchu, Japan
Correspondence
Koji Abe, Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2‐5‐1 Shikata‐cho, Kitaku, Okayama 700‐8558, Japan.
Email: [email protected]‐u.ac.jp Funding information
Grant‐in‐Aid for Scientific Research (C), Grant/Award Number: 15K0931607, 17H0419619 and 17K1082709; Grant‐in‐Aid for Scientific Research (B), Grant/Award Number: 17H0419619
Abstract
Stachybotrys microspora triprenyl phenol (SMTP)‐44D has both anti‐oxidative and anti‐inflammatory activities, but its efficacy has not been proved in relation to the pathological changes of neurovascular unit (NVU) and neurovascular trophic cou‐
pling (NVTC) in ischemic stroke. Here, the present study was designed to assess the efficacies of SMTP‐44D, moreover, compared with the standard neuroprotective reagent edaravone in ischemic brains. ICR mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min, SMTP‐44D (10 mg/kg) or edaravone (3 mg/kg) was intravenously administrated through subclavian vein just after the rep‐
erfusion, and these mice were examined at 1, 3, and 7 d after reperfusion. Compared with the vehicle group, SMTP‐44D treatment revealed obvious ameliorations in clini‐
cal scores and infarct volume, meanwhile, markedly suppressed the accumulations of 4‐HNE, 8‐OHdG, nitrotyrosine, RAGE, TNF‐α, Iba‐1, and cleaved caspase‐3 after tMCAO. In addition, SMTP‐44D significantly prevented the dissociation of NVU and improved the intensity of NAGO/BDNF and the number of BDNF/TrkB and BDNF/
NeuN double positive cells. These effects of SMTP‐44D in reducing oxidative and inflammatory stresses were similar to or stronger than those of edaravone. The pre‐
sent study demonstrated that SMTP‐44D showed strong anti‐oxidative, anti‐inflam‐
matory, and anti‐apoptotic effects, moreover, the drug also significantly improved the NVU damage and NVTC in the ischemic brain.
K E Y W O R D S
apoptosis, inflammatory, neurovascular unit, neurovascular trophic coupling, oxidative, SMTP‐44D, transient middle cerebral artery occlusion
1 | INTRODUCTION
Ischemic stroke is a major cause of mortality and death in the world.
Tissue plasminogen activator (t‐PA) is a thrombolytic enzyme, which had been proved as an effective treatment for acute ischemic stroke.
However, its narrow therapeutic time window (The National Institute of Neurological Disorders and Stroke rt‐PA Stroke Study Group, 1995) and risk of hemorrhagic transformation decreased the number of ben‐
eficiaries (The NINDS t‐PA Stroke Study Group, 1997). Thus, explor‐
ing new therapy is still a major challenge in the treatment of acute ischemic stroke.
SMTPs are a family of novel small molecules produced by the fun‐
gus Stachybotrys microspora (Hasumi, Yamamichi, & Harada, 2010).
Among SMTPs, SMTP‐7 was already reported to enhance plasmin‐
ogen activation through a conformational change in plasminogen, and effectively suppress oxidative/inflammatory stresses in ischemic stroke models (Akamatsu et al., 2011; Ito et al., 2014; Miyazaki et al., 2011; Shibata, Hashimoto, Hasumi, Honda, & Nobe, 2018; Shibata, Hashimoto, Nobe, Hasumi, & Honda, 2010). As well as SMTP‐7, an‐
other compound in the SMTPs family, SMTP‐44D (Koide, Hasegawa, Nishimura, Narasaki, & Hasumi, 2012), inhibits soluble epoxide hy‐
drolase (sEH) activity for lipid phosphate phosphatase at the N‐ter‐
minal domain and epoxide hydrolase at the C‐terminal domain (Matsumoto, Suzuki, Ishikawa, Shirafuji, & Hasumi, 2014). Unlike SMTP‐7, SMTP‐44D lacks a plasminogen modulation activity, but car‐
ries an antioxidative and anti‐inflammatory activities (Matsumoto et al., 2014). Anti‐inflammatory effect of SMTP‐44D was verified in ani‐
mal models of Guillain–Barré syndrome, ulcerative colitis and Crohn’s disease (Matsumoto et al., 2014) as well as in a model of thrombotic stroke (Shibata et al., 2018). However, the efficacy of SMTP‐44D in ischemic stroke has not been investigated in relation to neurovascular unit (NVU) and neurovascular trophic coupling (NVTC) as compared with edaravone.
In the present study, therefore, we evaluated the anti‐oxidative, anti‐inflammatory, and anti‐apoptotic efficacies of SMTP‐44D in relation to the changes of NVU damage and NVTC in transient
middle cerebral occlusion (tMCAO) mice model by comparing with edaravone.
2 | MATERIALS AND METHODS
2.1 | Animal
All experimental procedures were conducted according to the guide‐
lines of the Animal Care and Use Committee of the Graduate School of Medicine and Dentistry of Okayama University (OKU‐2016‐469).
Male ICR mice (7 weeks old, body weight 32–33 g; SLC, Shizuoka, Japan) were maintained in standard mouse cages under the con‐
trolled temperature around 23°C and housed with a 12/12 hr (h) light‐dark cycle for 2 weeks. Male mice were chosen because estro‐
gen may enhance neurogenesis and behavioral recovery following ischemic stroke in female mice (Koellhoffer & McCullough, 2013;
Suzuki, Brown, & Wise, 2009). One mouse was kept per cage. The mice were fasted from the day before the surgery around 12 at night and the total fasted time is less than 6 hr, but allowed free access to water.
2.2 | Experimental groups and drug treatments
Mice were divided randomly into four experimental groups: sham group, vehicle group (0.9% physiologic saline), SMTP‐44D group (10 mg/kg, TMS Co., Ltd., Tokyo, Japan) and edaravone group (3 mg/
kg, Mitsubishi Tanabe Pharma, Osaka, Japan). The drugs were ad‐
ministered by intravenous infusion (subclavian vein) immediately after reperfusion of tMCAO. Three sacrifice time points (1, 3, and 7 d after tMCAO) were divided into each drug administered group.
The investigators who performed the behavioral assessments, op‐
erations, and immunohistochemistry analysis were blinded to the treatment assignments.
2.3 | Focal cerebral ischemia
At the age of 8 weeks, the mice were anesthetized with a mixture of nitrous oxide/oxygen/halothane (69%:30%:1%) by an inhalation mask. An 8‐0 nylon filament thread with silicon coating was inserted through the right common carotid artery to occlude the right mid‐
dle cerebral artery (MCA) according to our previous reports (Abe, Kawagoe, Araki, Aoki, & Kogure, 1992; Yamashita et al., 2006). After 60 min of tMCAO, the nylon filament was softly pulled out to re‐
store blood flow of MCA. The successful artery occlusion was evalu‐
ated by measuring the decrease of regional cerebral flow of the right frontoparietal cortical region, before, during tMCAO and at reperfu‐
sion using Laser‐Doppler flowmeter (model ALF21; Advance, Tokyo, Japan). During this process, a heating pad (BWT‐100; Bio Research Center, Aichi, Japan) was used to monitor and maintain the body temperature at 37 ± 0.3°C. To detect an increase of infarct volume at different time point after surgery with a two‐sided 5% signifi‐
cance level and a power of 80%, a sample size of 5 mice per group Significance
An obvious anti‐inflammatory effect of SMTP‐44D was verified in ischemic animal models. Here, we evaluated the effects of SMTP‐44D on the neurovascular unit and neurovascular trophic coupling in ischemia. After 60 min of transient middle cerebral artery occlusion (tMCAO), SMTP‐44D showed strongly amelio‐
rated neurovascular unit and neurovascular trophic coupling damage, meanwhile, exerted significantly anti‐oxidative, anti‐in‐
flammatory, and anti‐apoptotic effects in ischemic mouse brains. Comparing with the standard neuroprotective reagent of edaravone, SMTP‐44D showed similar neuroprotective effects, which suggests a therapeutic potential of SMTP‐44D for human ischemic stroke patients in the future.
was necessary. We used 95 mice in this study, of which 40 were excluded based on the following exclusion criteria: mice that died as a result of a procedural problem during surgery (n = 12), mice with‐
out neurological findings (n = 8), mice that died after surgery in the period leading up to sacrifice (n = 20).
2.4 | Neurobehavioral and blood pressure analysis
For the evaluation of behavioral and blood pressure changes, same mice were tested before and on 1, 3 and 7 d after tMCAO. We as‐
sessed Bederson score (Bederson et al., 1986), with minor modifica‐
tions (Yamashita et al., 2009) as follows: 0, no observable neurologic deficits; 1, failure to extend the right forepaw; 2, circling to the con‐
tralateral side; 3, falling to the right; 4, unable to walk spontaneously.
On the same day, rotarod test was conducted with the same method
as our previous paper (MK610A; Muromachi Kikai Co., Tokyo, Japan) (Kawai et al., 2010; Ohta et al., 2008). Mice were placed on a rod and accelerated from 0 rpm to 45 rpm over a period of 5 min. The maximum time was recorded among three‐time tests and used as an indicator of the integrity of motor coordination. The corner test was performed (Li et al., 2004; Yu et al., 2005; Zhang et al., 2002) with slight modifications. The mouse was placed between two cardboard pieces (30 × 20 × 1 cm3), and encouraged to enter into a corner of 30° with a small opening along the joint between the two boards.
When the mouse entered into deep the part of the corner, both sides of the vibrissae were stimulated together by the two boards. Then, the mouse reared forward and upward, then turned back to face the open end. Ten trials were performed for each mouse and the number of right turns was calculated. Only turns involving full rearing along either board were recorded. Then calculated the difference value
TA B L E 1 Primary antibodies used
Antibody Description of immunogen Source, host species, catalog no., RRID
Concentration used
TNF‐α Escherichia coli‐derived recombinant
mouse TNF‐alpha
R and D Systems, goat polyclonal, AF‐410‐NA, AB_354479
1:100
Iba‐1 Synthetic peptide corresponding to
C‐terminus of Iba1
Wako, rabbit polyclonal, 019‐19741, AB_839504 1:1,000
Cleaved caspase‐3 Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino‐termi‐
nal residues adjacent to (Asp175) in human caspase‐3
Cell Signaling Technology, rabbit polyclonal, 9661,
AB_2341188 1:300
Nitrotyrosine Nitrated klh (keyhole limpet hemocyanin) Sigma, rabbit polyclonal, N0409, AB_260745 1:200
RAGE Synthetic peptide, (C)WRKRQP(R/L)
EERKAP ESQED(N), corresp. to aa 362‐380 of rat RAGE
Abcam, rabbit polyclonal, ab3611, AB_303947 1:200
4HNE Synthetic 4‐Hydroxynonenal modified
Keyhole Limpet Kemocyanin (KLH)
JalCA, mouse monoclonal, MHN‐100P, AB_1106813
1:50
8‐OHdG 8‐hydroxy‐2′‐deoxyguanosine is
produced by reactive oxygen and nitrogen species, including hydroxyl radical and peroxynitrite
JalCA, mouse monoclonal, MOG‐100P, AB_1106818
1:20
LEL LEL binds well to glycophorin and
Tamm‐Horsfall glycoprotein
Vector Laboratories, B‐1175, AB_2315475 1:200
NG2 HEK293 cells expressing a truncated
integral membrane form of NG2 consisting of amino acids 1592‐2222
Millipore, mouse monoclonal, 05‐710, AB_309925 1:1,000
GFAP Purified glial filament Abcam, goat polyclonal, ab53554, AB_880202 1:1,000
Collagen IV Full length native protein (extracted and purified from tumor tissues) (Mouse)
Abcam, rabbit polyclonal, ab19808, AB_445160 1:500
BDNF A synthetic peptide from human mature
BDNF conjugated to an immunogenic carrier protein
Abcam, sheep polyclonal, ab75040, AB_1280756 1:100
TrkB A peptide mapping within a C‐terminalcy‐
toplasmic domain of Trk B of mouse origin
Santa Cruz Biotechnology, rabbit polyclonal, sc‐8316, AB_2155274
1:100
NeuN Purified cell nuclei from mouse brain Millipore, mouse monoclonal, MAB377, AB_2298772
1:200
before and after the operation. This task detected integrated sen‐
sorimotor function as it involves both stimulations of the vibrissae (sensory/neglect) and rearing (motor response).
Blood pressure was measured by the method of tail‐cuff with heating (BP‐98A; Softron, Tokyo, Japan). Mice were preheated in a chamber at 37°C for 10 min, then placed in restrainers. A cuff with a pneumatic pulse sensor was attached to the tail. Blood pressure values were showed on the monitor.
2.5 | Tissue preparation
Before sacrifice in each time point, the survival mice (total n = 55; 5 for sham group, 16 for vehicle group, 17 for SMTP‐44D group, 17 for edaravone group) were anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg) and transcardially perfused with chilled phosphate‐buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). Then, the whole brain was removed and immersed in the same fixation overnight at 4°C. After washing with PBS, the fixed tissues were transferred into 10%, 20%, and 30% (wt/vol) sucrose in PBS, each sucrose incubation step was for 24 hr at 4°C. Then, the brains were frozen in dry ice and kept at
−80°C. Successive 20‐μm thick coronal sections were prepared on a cryostat at −18°C and mounted on silane‐coated glass slides.
2.6 | Infarct volume measurement
Coronal sections were stained with cresyl violet as Nissl staining and examined with a light microscope (Olympus BX‐51; Olympus Optical, Tokyo, Japan) to analyze the infarct volume. The border between the ischemic core and peri‐infarct lesion were confirmed through Nissl staining of adjacent sections as reported earlier (Omori et al., 2002).
The infarct area was measured in five sections by counting pixels using Photoshop 7.0 (1 mm2~1,145.8 pixels in this experiment), and the infarct volume of each brain was calculated by summation of in‐
farct areas of five serial brain slices, at a 0.5 mm interval each, be‐
tween 1.0 mm anterior and 1.5 mm posterior to the bregma (Nakano et al., 2017; Shang, Liu, Tanaka, & Abe, 2012).
2.7 | Single immunohistochemistry analysis
Brain sections were first washed three times in PBS. To block intrinsic peroxidase activities, we used 0.3% hydrogen peroxide/methanol for 30 min. To block non‐specific antibody expressions, sections were dealt with 5% bovine serum albumin in PBS with 0.1% triton for 1 hr. Slides were incubated at 4°C overnight with the following primary antibodies (see Table 1): goat antitumor necrosis factor alpha (TNF‐α) antibody (1:100; R and D Systems, Minneapolis, MN, AF‐410‐NA, AB_354479);
rabbit anti‐ionized calcium‐binding adapter molecule 1 (Iba‐1) anti‐
body (1:1,000; Wako, Osaka, Japan, 019‐19741, AB_839504); rabbit anti‐cleaved caspase‐3 antibody (1:300; Cell Signaling Technology, Beverly, MA, 9661, AB_2341188); rabbit anti‐nitrotyrosine antibody (1:200; Sigma, St. Louis, MO, N0409, AB_260745) and rabbit anti‐re‐
ceptor for advanced glycation end products (RAGE) antibody (1:200;
Abcam, Cambridge, MA, ab3611, AB_303947). Brain sections were then washed with PBS and treated with suitable biotinylated second‐
ary antibodies (1:500; Vector Laboratories, Burlingame, CA) for 2 hr at room temperature. Immunohistochemistry for mouse anti‐4‐hy‐
droxy‐2‐nonenal (4‐HNE) (1:50; JalCA, Shizuoka, Japan, MHN‐100P, AB_1106813) and mouse anti‐8‐hydroxy‐2′‐deoxyguanosine (8‐
OHdG) (1:20; JalCA, Shizuoka, Japan, MOG‐100P, AB_1106818) were performed with mouse on mouse immunodetection kit (Vector Laboratories) (Kusaki et al., 2017). The slides were then treated with avidin‐biotin‐peroxidase complex (Vectastain ABC Kit; Vector) for 30 min and incubated with diaminobenzidine tetrahydrochloride. As for the negative control, we stained a set of brain sections in the same manner without the primary antibody. A light microscope (Olympus BX‐51, Tokyo, Japan) was used to examine the sections.
2.8 | Double‐fluorescence immunohistochemistry
The damage of NVU and the changes of NVTC were ascertained by double immunofluorescence staining for N‐acetylglucosamine oligom‐
ers (NAGO) plus neuron‐glial antigen 2 (NG2), NG2 plus glial fibrillary acidic protein (GFAP), collagen IV plus GFAP, NAGO plus brain‐derived neurotrophic factor (BDNF), BDNF plus tropomyosin receptor kinase B (TrkB) and BDNF plus neuronal nuclear antigen (NeuN). NAGO is used as a specific marker expressed in mature vascular endothelial cells, while lycopersicon esculentum lectin (LEL) is a glycoprotein with positive af‐
finity for NAGO (Augustin, Braun, Telemenakis, Modlich, & Kuhn, 1995).
The following primary antibodies were used: biotinylated LEL (1:200;
Vector, B‐1175, AB_2315475); mouse anti‐NG2 antibody (1:1,000;
Millipore, Burlington, MA, 05‐710, AB_309925); goat anti‐GFAP anti‐
body (1:1,000; Millipore, ab53554, AB_880202); rabbit anti‐collagen IV antibody (1:500; Abcam, ab19808, AB_445160); sheep anti‐BDNF antibody (1:100; Abcam, ab75040, AB_1280756); rabbit anti‐TrkB an‐
tibody (1:100; Santa Cruz Biotechnology, CA, sc‐8316, AB_2155274);
mouse anti‐NeuN antibody (1:200; Millipore, Burlington, MA, MAB377, AB_2298772). Then immunoreactions were visualized using fluores‐
cent secondary antibody or horseradish peroxidase‐conjugated an‐
tibody with diaminobenzidine reaction. The treated brain sections were scanned with a confocal microscope equipped with an argon and HeNe1 laser (LSM‐510; Zeiss, Jena, Germany).
2.9 | Vascular dissociation index
Detachment of pericytes from vascular endothelial cells and astro‐
cyte endfeet, and that of basement membrane from astrocyte end‐
feet were evaluated by discriminating stainings of NAGO/NG2, NG2/
GFAP and collagen IV/GFAP double labeled sections. The treated sections were evaluated by randomly selecting three levels of the striatum and four areas in the ipsilateral peri‐infarcted cortex in each section (Yamashita et al., 2009). We confirmed the border between the ischemic core and peri‐infarcted lesion using cresyl violet stain‐
ing of adjacent sections according to a previous method (Omori et al., 2002). We measured the area between the astrocyte endfeet and basement membrane of each blood vessel, and the length of each
blood vessel. Then, the area‐to‐length ratio was calculated as the vascular dissociation index (Yamashita et al., 2009). In a similar way, the area between pericytes and vascular endothelial cells and the area between astrocyte endfeet and pericytes were calculated, and the area‐to‐length ratio was measured of each blood vessel.
2.10 | Semiquantitative analysis
We used the method of semiquantitative analysis to evaluate the changes of immunohistochemical staining in TNF‐α, Iba‐1, cleaved caspase‐3, 4HNE, 8‐OHdG, nitrotyrosine, and RAGE. In the peri‐ischemic lesion of each section, we calculated three sepa‐
rated sections per brain and five random selected regions. In the same method, we evaluated the intensity of NAGO/BDNF staining using an image analysis software (Scion Image, Scion Corporation, Frederick, MD, SCR_008673). For the quantification of BDNF/
TrkB and BDNF/NeuN double positive cells, we counted the num‐
ber of double positive cells in the peri‐infarcted cortex (cells/mm2).
2.11 | Statistical analysis
Data were analyzed in GraphPad Prism (version 7.0, GraphPad Software Inc., San Diego, CA, SCR_002798) and presented as
mean ± SD or median (interquartile range). Two‐way analysis of ANOVA was used to examine the differences in the neurobehav‐
ioral analysis and the expression of single immunohistochemistry analysis between each drug treated group and time points followed by Tukey’s multiple comparisons test and Dunnett’s multiple com‐
parisons test. One‐way analysis of ANOVA was performed to verify the differences in the expression of double fluorescence immuno‐
histochemistry followed by a Dunnett’s multiple comparisons test.
In all statistical analyses, significance was accepted at p < 0.05.
3 | RESULTS
3.1 | Clinical scores and infarct volume after tMCAO
As compared to the vehicle group, SMTP‐44D treatments sig‐
nificantly improved the Bederson’s scores at 7 d after tMCAO (time F3,60 = 62, p < 0.0001; treatment F2,60 = 4.083, p = 0.0217;
interaction F6,60 = 0.75, p = 0.6118; total n = 54, 18 mice at each time point;*p < 0.05 vs. vehicle group; Figure 1a). Corner test scores significantly improved at 7 d compared to the scores at 1 d after SMTP‐44D and edaravone treatments (time F2,45 = 6.41, p = 0.0036; treatment F2,45 = 2.352, p = 0.1067; interaction F4,45 = 0.4095, p = 0.8008; total n = 54, 18 mice at each time
F I G U R E 1 Clinical scores of mice after tMCAO. Bederson score (a), corner test (b), SBP (c), DBP (d), MBP (e), and rotarod time (f) at 1, 3, and 7 d after tMCAO in the vehicle, SMTP‐44D and edaravone groups. *p < 0.05 versus vehicle group; #p < 0.05 versus 1 d
point; #p < 0.05 vs. 1 d; Figure 1b). In the result of blood pressure, SMTP‐44D treatments significantly decreased systolic, diastolic and mean blood pressure (SBP, DBP and MBP) compared with the value at 1 d (SBP: time F2,42 = 7.239, p = 0.0020; treatment F2,42 = 2.057, p = 0.1406; interaction F4,42 = 0.4956, p = 0.7390;
DBP: time F2,42 = 7.059, p = 0.0023; treatment F2,42 = 3.293, p = 0.0470; interaction F4,42 = 0.3436, p = 0.8469; MBP: time F2,42 = 8.187, p = 0.0010; treatment F2,42 = 3.575, p = 0.0368; in‐
teraction F4,42 = 0.3408, p = 0.8489; total n = 51, 17 mice at each time point; #p < 0.05 vs. 1 d; Figure 1c–e). Both SMTP‐44D and edaravone treatments significantly improved the rotarod time at 7 d compared with the time at 1 d (time F2,45 = 10.18, p = 0.0002;
treatment F2,45 = 5.172, p = 0.0095; interaction F4,45 = 0.3197, p = 0.8633; total n = 54, 18 mice at each time point; #p < 0.05 vs. 1 d; Figure 1f).The infarct volumes after tMCAO increased at 3 d and significantly decreased at 7 d compared to 1 and 3 d in all three groups, both SMTP‐44D and edaravone treatments displayed sig‐
nificant differences at 1, 3, and 7 d compared with vehicle (time F2,41 = 70.36, p < 0.0001; treatment F2,41 = 26.44, p < 0.0001;
interaction F4,41 = 0.7566, p = 0.5595; total n = 50; *p < 0.05 and
**p < 0.01 vs. vehicle group, ##p < 0.01 vs. 1 d, §§p < 0.01 vs. 3 d;
Figure 2a,b).
3.2 | SMTP‐44D and edaravone treatments reduced oxidative stress
At 1 d after tMCAO, 4‐HNE, 8‐OHdG, nitrotyrosine, and RAGE were strongly expressed in the peri‐infarct cerebral cortex, which were ameliorated in SMTP‐44D and edaravone groups (Figure 3a,c,e,g).
The number of 4‐HNE positive cells significantly increased at 3 d compared to 1 d in the vehicle and edaravone group (Figure 3a,b,
#p < 0.05 and ##p < 0.01 vs. 1 d), but significantly decreased at 7 d compared to 3 d after tMCAO in vehicle, SMTP‐44D and edaravone groups (Figure 3a,b, §§p < 0.01 vs. 3 d). Compared with the vehicle group, the number of 4HNE positive cells were significantly de‐
creased in the sham, SMTP‐44D and edaravone groups at 1, 3, and 7 d (time F2,53 = 26.03, p < 0.0001; treatment F3,53 = 194.7, p < 0.0001;
interaction F6,53 = 4.704, p = 0.0007; total n = 55; *p < 0.05 and
**p < 0.01 vs. vehicle group; Figure 3a,b).
The number of 8‐OHdG positive cells significantly increased at 3 d compared to 1 d in the vehicle and edaravone group (Figure 3c,d,
##p < 0.01 vs. 1 d), but significantly decreased at 7 d compared to 3 d in vehicle, SMTP‐44D and edaravone groups (Figure 3c,d, §§p < 0.01 vs. 3 d). Compared with the vehicle group, SMTP‐44D treatment significantly decreased the number of 8‐OHdG positive cells at 1 and 3 d, while edaravone treatment significantly decreased the number of positive cells at 3 d (time F2,53 = 38.18, p < 0.0001; treat‐
ment F3,53 = 102.7, p < 0.0001; interaction F6,53 = 5.065, p = 0.0004;
**p < 0.01 vs. vehicle group; Figure 3c,d).
The number of nitrotyrosine positive cells significantly in‐
creased at 3 and 7 d compared to 1 and 3 d in vehicle, SMTP‐44D and edaravone groups (Figure 3e,f, #p < 0.05 and ##p < 0.01 vs. 1 d,
§p < 0.05 and §§p < 0.01 vs. 3 d). Compared with the vehicle group, SMTP‐44D treatment significantly decreased the number of nitroty‐
rosine positive cells at 1, 3, and 7 d, while edaravone treatment sig‐
nificantly decreased the number of positive cells only at 3 and 7 d (time F2,53 = 52.21, p < 0.0001; treatment F3,53 = 269.6, p < 0.0001;
interaction F6,53 = 6.136, p < 0.0001; **p < 0.01 vs. vehicle group;
Figure 3e,f).
The numbers of RAGE positive cells significantly increased at 3 and 7 d compared to 1 and 3 d in vehicle, SMTP‐44D and eda‐
ravone groups (Figure 3g,h, ##p < 0.01 vs. 1 d, §§p < 0.01 vs. 3 d).
SMTP‐44D and edaravone treatments significantly decreased the number of RAGE positive cells compared with vehicle at 3 and 7 d (time F2,53 = 120.5, p < 0.0001; treatment F3,53 = 129.6, p < 0.0001;
interaction F6,53 = 15.53, p < 0.0001; **p < 0.01 vs. vehicle group;
Figure 3g,h).
3.3 | SMTP‐44D and edaravone treatments improved inflammatory changes and apoptosis
After tMCAO, the number of TNF‐α positive cells significantly de‐
creased in the peri‐infarct cortex at 3 and 7 d compared to 1 d in vehicle and edaravone group, and at 7 d compared to 1 and 3 d
F I G U R E 2 Ischemic infarct volumes of mice after tMCAO. (a) Nissl stainings of mice brain at 1, 3 and 7 d after tMCAO in the vehicle, SMTP‐44D and edaravone group. (b) The quantitative analysis of infarct volumes in the three groups. *p < 0.05; **p < 0.01 versus vehicle group, ##p < 0.01 versus 1 d, §§p < 0.01 versus 3 d [Colour figure can be viewed at wileyonlinelibrary.com]
in SMTP‐44D group (Figure 4a,b, #p < 0.05 and ##p < 0.01 vs. 1 d,
§p < 0.05 vs. 3 d). Compared with the vehicle group, the number of TNF‐α positive cells were significantly decreased in the sham, SMTP‐44D and edaravone groups at 1, 3 and 7 d (time F2,53 = 29.05, p < 0.0001; treatment F3,53 = 186.2, p < 0.0001; interaction
F6,53 = 3.342, p = 0.0072; total n = 55; *p < 0.05 and **p < 0.01 vs. ve‐
hicle group; Figure 4a,b).
After tMCAO, the number of Iba‐1 positive cells significantly in‐
creased in the peri‐infarct cortex at 3 and 7 d compared to 1 and 3 d in vehicle and SMTP‐44D group, and at 7 d compared to 1 and 3 F I G U R E 3 The expression of oxidative stress markers in the ischemic mice brain. Single immunohistochemical stainings of 4‐HNE (a), 8‐
OHdG (c), nitrotyrosine (e) and RAGE (g) in the peri‐ischemic area at 1, 3, and 7 d after tMCAO of sham, vehicle, SMTP‐44D and edaravone groups. Scale bar = 50 μm. Quantitative analysis of the number of 4‐HNE (b), 8‐OHdG (d), nitrotyrosine (f) and RAGE (h) positive cells in the peri‐ischemic area of the four groups after tMCAO. *p < 0.05; **p < 0.01 versus vehicle group, #p < 0.05; ##p < 0.01 versus 1 d, §p < 0.05;
§§p < 0.01 versus 3 d [Colour figure can be viewed at wileyonlinelibrary.com]
d in edaravone group (Figure 4c,d, ##p < 0.01 vs. 1 d, §§p < 0.05 vs.
3 d). Compared with the vehicle group, SMTP‐44D treatment sig‐
nificantly decreased the number of positive cells at both 1 and 7 d, while edaravone treatment significantly decreased the number of Iba1 positive cells at 3 and 7 d (time F2,53 = 55.52, p < 0.0001; treat‐
ment F3,53 = 107, p < 0.0001; interaction F6,53 = 6.467, p < 0.0001;
*p < 0.05 and **p < 0.01 vs. vehicle group; Figure 4c,d).
After tMCAO, the number of cleaved caspase‐3 positive cells significantly decreased at 7 d compared to 1 and 3 d in vehicle, SMTP‐44D and edaravone groups (Figure 4e,f, ##p < 0.01 vs. 1 d,
§p < 0.05 and §§p < 0.05 vs. 3 d). Compared with the vehicle group, SMTP‐44D treatment significantly decreased the number of posi‐
tive cells at 1, 3, and 7 d, while edaravone treatment significantly de‐
creased the number of cleaved caspase‐3 positive cells at 1 and 7 d (time F2,53 = 20.95, p < 0.0001; treatment F3,53 = 65.24, p < 0.0001;
interaction F6,53 = 2.265, p = 0.0509; *p < 0.05 and **p < 0.01 vs. ve‐
hicle group; Figure 4e,f).
3.4 | NVU and NVTC
The vascular dissociation index revealed that there was no difference among the three groups in terms of dissociation between vascular endothelial cells (NAGO) and pericytes (NG2) at 1 d after tMCAO (F3,15 = 0.5654, p = 0.6461; n = 19; Figure 5a,d). However, significant dissociations were observed between pericytes (NG2) and astro‐
cyte foot processes (GFAP), and between basal lamina (collagen IV) and GFAP in the vehicle group, which were significantly improved in sham, SMTP‐44D and edaravone treated groups (F3,15 = 14.93, p < 0.0001; F3,15 = 10.68, p = 0.0005; *p < 0.05 and **p < 0.01 vs. ve‐
hicle group; Figure 5b,c,e,f).
F I G U R E 4 The expression of inflammatory and apoptotic markers in the ischemic mice brain. Single immunohistochemical stainings of TNF‐α (a), Iba‐1 (c) and cleaved caspase‐3 (e) in the peri‐ischemic area at 1, 3, and 7 d after tMCAO of sham, vehicle, SMTP‐44D and edaravone groups. Scale bar = 50 μm. Quantitative analysis of the number of TNF‐α (b), Iba‐1 (d) and cleaved caspase‐3 (f) positive cells in the peri‐ischemic area of the four groups after tMCAO. *p < 0.05; **p < 0.01 versus vehicle group, #p < 0.05; ##p < 0.01 versus 1 d, §p < 0.05;
§§p < 0.01 versus 3 d [Colour figure can be viewed at wileyonlinelibrary.com]
Double‐fluorescent intensity of NAGO/BDNF decreased in the vehicle group at 1 d after tMCAO compared with sham (F3,15 = 4.817, p = 0.0152; n = 19; **p < 0.01 vs. vehicle group; Figure 6a,d).
SMTP‐44D and edaravone treatments significantly improved the
intensity compared with vehicle (Figure 6a,d, *p < 0.05 vs. vehicle group). The significant reduction of BDNF/TrkB double positive cell numbers in the vehicle group which compared with sham were sig‐
nificantly recovered in the SMTP‐44D and edaravone treatments F I G U R E 5 Double‐fluorescence immunohistochemical stainings showing NVU damage with NAGO/NG2 (a), NG2/GFAP (b) and
collagen IV/GFAP (c) in the peri‐ischemic area at 1 d after tMCAO of sham, vehicle, SMTP‐44D and edaravone groups. Scale bar = 20 μm.
Quantitative vascular dissociation index of NAGO/NG2 (d), NG2/GFAP (e) and collagen IV/GFAP (f) at 1 d after tMCAO of the four groups.
Note the significantly improved vascular dissociation indexes of NG2/GFAP (E) and collagen IV/GFAP (f) in SMTP‐44D and edaravone treated groups compared with vehicle group. *p < 0.05; **p < 0.01 versus vehicle group [Colour figure can be viewed at wileyonlinelibrary.
com]
(F3,15 = 6.01, p = 0.0067; n = 19; *p < 0.05 and **p < 0.01 vs. vehicle group; Figure 6b,e), as well as BDNF/NeuN double positive cells (F3,15 = 7.356, p = 0.0029; *p < 0.05, and **p < 0.01 vs. vehicle group;
Figure 6c,f).
4 | DISCUSSION
In the present study, we demonstrated that SMTP‐44D treatment sig‐
nificantly improved the neurobehaviors of mice (Figure 1), decreased F I G U R E 6 Double‐fluorescence immunohistochemical stainings showing neurovascular tropic coupling change with NAGO/BDNF (a), BDNF/TrkB (b) and BDNF/NeuN (c) in the peri‐ischemic area at 1 d after tMCAO of sham, vehicle, SMTP‐44D and edaravone groups. Scale bar = 50 μm. (d) Quantitative fluorescent intensity of NAGO/BDNF at 1 d after tMCAO of the four groups. Double positive cells of BDNF/
TrkB (e) and BDNF/NeuN (f) after tMCAO of the four groups. *p < 0.05; **p < 0.01 versus vehicle group [Colour figure can be viewed at wileyonlinelibrary.com]
infarct volumes, oxidative stress and inflammatory changes (Figures 2‒4), and prevented apoptosis and damage to NVU and NVTC in the mice brain after tMCAO (Figures 4‒6). These effects of SMTP‐44D were similar to the standard neuroprotective reagent edaravone for NVU and NVTC damage (Figures 5 and 6), or even stronger than edaravone in reducing oxidative, inflammatory, and apoptotic stresses (Figures 2 and 3).
Unlike edaravone, SMTP‐44D treatment significantly de‐
creased blood pressure of mice after tMCAO (Figure 1c), prob‐
ably due to the primary action of SMTP‐44D for inhibiting sEH (Matsumoto et al., 2014; Sinal et al., 2000; Yu et al., 2000).
Transient cerebral ischemia increases oxidative stress after reper‐
fusion, and edaravone has strong neuroprotective effects by reducing free radical damage to the ischemic brain (Abe, 2000;
Abe, Yuki, & Kogure, 1988; Chan, 1996; Morimoto, Globus, Busto, Martinez, & Ginsberg, 1996; Yamashita et al., 2009; Zhang et al., 2004). In addition to the previous report describing SMTPs’ activ‐
ities to inhibit lipid peroxidation (Shibata et al., 2018), the present study newly showed that SMTP‐44D reduced peroxidative dam‐
ages of lipids (4‐HNE), DNA (8‐OHdG), proteins (nitrotyrosine) and sugar (RAGE) (Figure 3) (Park, Yun, & Park, 2009; Shang et al., 2016; Takeuchi et al., 2000).
The present study also showed a strong anti‐inflammatory effect of SMTP‐44D in the ischemic mice brain (Figure 4). Among the SMTP family, SMTP‐7 have thrombolytic and anti‐inflammatory effects in the ischemic mice brain (Hashimoto, Shibata et al., 2010; Sawada et al., 2014; Shibata et al., 2010). On the other hand, SMTP‐44D lacks such a fibrinolytic activity but exerts a strong anti‐inflammatory, anti‐oxidative, and anti‐apoptotic activities (Figures 3 and 4), which finally reduced infarct volume of tMCAO mice model (Figure 2).
The present study first showed that SMTP‐44D ameliorated NVU dissociation between pericyte and astrocytic foot processes, and between basal lamina and astrocytic foot processes (Figure 5).
TrkB is an endogenous receptor of BDNF, and BDNF establishes a trophic link between cerebral endothelium and neuronal survival (Guo et al., 2008). The present study showed that SMTP‐44D and edaravone similarly improved the damage to endothelial neuropro‐
tective support for the outsider neurons in the stainings of NAGO/
BDNF, BDNF/TrkB, and BDNF/NeuN (Figure 6).
SMTP‐44D inhibits the effect of sEH converting from epoxye‐
icosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (Matsumoto et al., 2014). Previous papers showed that EETs had anti‐oxidative (Yang et al., 2001), anti‐inflammatory (Imig, 2012), anti‐apoptotic (Yang et al., 2007), vasodilatory (Ellis, Amruthesh, Police, & Yancey, 1991), angiogenic (Zhang & Harder, 2002), fibrinolyic (Node et al., 2001) and anti‐thrombotic (Heizer, McKinney, & Ellis, 1991) effects.
In addition, the epoxyeicosanoid signaling worked protectively on NVU in rat ischemic model (Liu et al., 2016). We speculated that EETs might be the target of neuroprotective effect of SMTP‐44D.
In summary, the present study first showed the strong anti‐
oxidative, anti‐inflammatory, and anti‐apoptotic effects of SMTP‐44D, which also significantly improved NVU damage and NVTC in the ischemic brain similar to edaravone. Significant
improvement in neurologic deficit scores and infarct volumes sug‐
gests a stronger therapeutic potential of SMTP‐44D compared with edaravone for human ischemic stroke patients in the future.
ACKNOWLEDGMENTS
This work was partly supported by a Grant‐in‐Aid for Scientific Research (B) 17H0419619, (C) 15K0931607, 17H0419619 and 17K1082709, and by Grants‐in‐Aid from the Research Committees (Kaji R, Toba K, and Tsuji S) from Japan Agency for Medical Research and development (AMED).
CONFLIC T OF INTEREST
The authors disclose no potential conflict of interests.
AUTHOR CONTRIBUTIONS
All authors had full access to all the data in the study and take re‐
sponsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: K.A. Acquisition of data: X.S., J.S.
Analysis and interpretation of data: X.S., J.S., X.L. Drafting of the manu‐
script: X.S. Critical revision of the manuscript for important intellectual content: Y.O., J.S., T.Y., E.S., K.H., K.A. Statistical analysis: X.S., J.S.
Obtained funding: K.A., Y.O., T.Y. Administrative, technical, and mate‐
rial support: X.S., J.S., R.M., Y.N., Y.F., X.L., T.F., Y.H., K.S., M.T., N.H., E.S., K.H. Study supervision: K.A.
ORCID
Xiaowen Shi http://orcid.org/0000‐0001‐8806‐8226
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SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Shi X, Ohta Y, Shang J, et al.
Neuroprotective effects of SMTP‐44D in mice stroke model in relation to neurovascular unit and trophic coupling. J Neuro Res. 2018;96:1887–1899. https://doi.org/10.1002/jnr.24326
