Receptor for Advanced Glycation End Products Is Involved in LPA5-Mediated Brain Damage after a Transient Ischemic Stroke

Lysophosphatidic acid receptor 5 (LPA5) has been recently identified as a novel pathogenic factor for brain ischemic stroke. However, its underlying mechanisms remain unclear. Here, we determined whether the receptor for advanced glycation end products (RAGE) could be involved in LPA5-mediated brain injuries after ischemic challenge using a mouse model of transient middle cerebral artery occlusion (tMCAO). RAGE was upregulated in the penumbra and ischemic core regions after tMCAO challenge. RAGE upregulation was greater at 3 days than that at 1 day after tMCAO challenge. It was mostly observed in Iba1-immunopositive cells of a post-ischemic brain. Suppressing LPA5 activity with its antagonist, TCLPA5, attenuated RAGE upregulation in the penumbra and ischemic core regions, particularly on Iba1-immunopositive cells, of injured brains after tMCAO challenge. It also attenuated blood–brain barrier disruption, one of the core pathogenesis upon RAGE activation, after tMCAO challenge. As an underlying signaling pathways, LPA5 could contribute to the activation of ERK1/2 and NF-κB in injured brains after tMCAO challenge. Collectively, the current study suggests that RAGE is a possible mediator for LPA5-dependent ischemic brain injury.


Introduction
Lysophosphatidic acid (LPA) is an important bioactive lipid that can regulate various biological functions by activating its specific six G protein-coupled receptors (LPA [1][2][3][4][5][6] [1,2]. LPA receptors are known to play critical roles in the pathogenesis of various diseases [2][3][4]. Thus, several efforts have been made to develop drugs by targeting LPA signaling including its receptors [2][3][4]. In brain ischemic stroke, the importance of receptor-mediated LPA signaling has been suggested [5][6][7][8][9][10]. Amounts of LPA can be increased in both human patients [5] and animals with stroke [6,7]. Moreover, its receptors LPA 1 [8] and LPA 5 [9,10] have been demonstrated to contribute to ischemic brain injuries. In particular, LPA 5 has been suggested to play a pathogenic role in brain ischemic stroke because it can be upregulated in the ischemic core regions after ischemic challenge [9] and suppressing its activity with TCLPA5, a selective LPA 5 antagonist, can lead to neuroprotection against ischemic brain injuries during both acute and chronic phases [9,10]. However, how LPA 5 can play its pathogenic role remains unclear. The receptor for advanced-glycation end products (RAGE) is a multiligand receptor that can bind to advanced glycation end products (AGEs), amyloid beta (Aβ), the S100/calgranulin family, and high mobility group box 1 non-histone DNA-binding protein (HMGB1 or amphoterin) [11]. It has been reported that RAGE plays various pathological roles in regulating cytokines production, Aβ accumulation, protein aggregation, and immune cell infiltration in brain disorders, such as Alzheimer disease, intracerebral hemorrhage (ICH), multiple sclerosis, and brain ischemic stroke [11][12][13]. In particular, in brain ischemic stroke, RAGE can be upregulated in injured brains and either pharmacological or genetic suppression of RAGE activities can attenuate brain injuries [14][15][16]. Such Life 2021, 11, 80 2 of 12 pathogenic roles of RAGE can be associated with proinflammatory cytokine production, NF-κB activation, and gliosis [14][15][16]. In view of the link between RAGE and LPA signaling, it has been previously suggested that RAGE can mediate LPA-dependent biological functions in cancer, such as cancer cell growth in vitro and tumor formation in vivo [17,18]. However, the possible relationship between RAGE and LPA signaling in brain ischemic stroke is unexplored. Moreover, whether LPA receptors could be involved in such a relationship remains unknown.
Here, we addressed whether RAGE could be involved in LPA 5 -mediated pathogenesis of post-ischemic brains with a mouse model of transient middle cerebral artery occlusion (tMCAO). We determined whether LPA 5 could regulate RAGE expression in injured brains after tMCAO challenge using an LPA 5 antagonist, TCLPA5. We also determined whether LPA 5 could contribute to blood-brain barrier (BBB) disruption, one of core pathogenesis regulated by RAGE [15,16,19]. Finally, we determined whether LPA 5 could contribute to the activation of ERK1/2 and NF-κB, both of which could be effector pathways of RAGE to mediate BBB disruption.

Animals
Male ICR mice were purchased from Orient Bio (Seongnam-Si, Korea). All of the animal experiments were carried out following Institutional Animal Care and Use guidelines of Lee Gil Ya Cancer and Diabetes Institute (LCDI) at Gachon University (approved animal protocol number: LCDI-2019-0027). Mice were housed under a controlled environment with a light cycle of 12 h/12 h of day/night, temperature of 22 ± 2 • C, and a relative humidity of 60 ± 10%. Mice had free access to food and water.

tMCAO Challenge
ICR mice (male, 7 weeks old) were challenged with tMCAO as described previously [9]. Briefly, mice were anesthetized with isoflurane in a mixture of O 2 :N 2 O (1:3) (3% for induction and 1.5% for maintenance). A ventral neck incision was made and the right common carotid artery (CCA) was carefully separated from the vagus nerve. MCAO was induced by inserting a monofilament suture coated with silicon (9 mm long, 5-0) towards the MCA through an internal carotid artery from CCA bifurcation. At 90 min after occlusion, the monofilament was withdrawn to restore the blood flow under an anesthetized condition. For the sham group, mice were subjected to the same surgical procedure except MCAO. Body temperature was maintained at 37 • C during surgery.

Vehicle or TCLPA5 Administration
Mice subjected to the MCAO surgery were randomly divided into a vehicle (10% Cremophor EL and 10% ethanol in distilled water)-administered group or a TCLPA5administered group. TCLPA5 (5-(3-Chloro-4-cyclohexylphenyl)-1-(3-methoxyphenyl)-1Hpyrazole-3-carboxylic acid, 10 mg/kg, i.p. Tocris Bioscience, Bristol, UK) was administered to mice immediately after reperfusion. TCLPA5 dose was selected based on our previous study [9]. Two mice of the vehicle-administered group were excluded from the study because of death. In the TCLPA5-administered group, three mice were excluded from the study because of death (two mice) or hemorrhage (one mouse). The number of mice per group was 4, 5, and 5 for Western blot, histological, and quantitative real-time PCR (qRT-PCR) analyses, respectively.

RAGE Immunohistochemistry
Brain sections were post-fixed with 4% PFA, treated with 0.01 M sodium citrate at 90-100 • C, oxidized with 1% H 2 O 2 , and blocked with 1% fetal bovine serum (FBS) in PBS containing 0.3% Triton X-100. Brain sections were labelled with mouse anti-RAGE (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 • C followed by an incubation with a biotinylated secondary antibody (1:200, Santa Cruz Biotechnology) for 2 h at room temperature. Tissue sections were then incubated with an ABC reagent (1:100, Vector Laboratories, Burlingame, CA, USA) for 90 min at room temperature. Signals were developed using a 3,3 -diaminobenzidine (DAB) kit (Dako, Santa Clara, CA, USA) for 2 min, washed with water, dehydrated with alcohol and xylene, and mounted with Entellan media (Merck, Darmstadt, Germany).

Image Preparation and Quantification
Brain images were taken from either a bright-field microscope equipped with a DP72 camera (BX53T, Olympus, Japan) or a laser scanning confocal microscope (Eclipse A1 Plus, Nikon, Japan). Representative images were prepared with Adobe Photoshop Elements 8 (Adobe Inc., San Jose, CA, USA). Three different images (600 µm × 600 µm) of each brain region in a mouse were used to quantify numbers of immunopositive cells. Data are provided as mean value of these numbers of immunopositive cells per unit area (mm 2 ).

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). Date are presented as mean ± S.E.M. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by a Newman-Keuls post-hoc test for comparisons among groups. Statistical significance was considered when p value was less than 0.05.

RAGE Is Upregulated in the Penumbra and Ischemic Core Regions of Injured Brains, Particularly on Iba1-Positive Cells, after tMCAO Challenge
RAGE can be upregulated in human ischemic patients [20] and animals with brain ischemic stroke [15,16,20]. It has been also previously reported that RAGE upregulation can occur in the penumbra region after a permanent brain ischemic stroke [20]. In the current study, we determined region-and time-specific RAGE upregulation by immunohistochemical analysis at 1 day and 3 days after tMCAO challenge. The number of RAGE-positive cells was significantly increased at both 1 day and 3 days after tMCAO challenge compared to that in the sham group (Figure 1a,b). RAGE upregulation was observed in the penumbra and ischemic core regions, but not in the periischemic regions (Figure 1a,b). The degree of RAGE immunoreactivities seemed to be stronger in the ischemic core region than in the penumbra (Figure 1a,b). In addition, the number of RAGE-positive cells was higher at 3 days after tMCAO challenge than that at 1 day after tMCAO challenge ( Figure 1a,b). These results indicate that RAGE could be upregulated in lesional areas like the penumbra and ischemic core regions after an ischemic stroke.
Next, we determined in which cell types RAGE upregulation could occur at 3 days after tMCAO challenge by double immunofluorescence staining for RAGE with cell markers Iba1 (activated microglia/macrophage), GFAP (activated astrocyte), NeuN (neuron), and CD31 (endothelial cell). The number of RAGE/Iba1-, RAGE/NeuN-, and RAGE/CD31double immunopositive cells was analyzed in the ischemic core regions because RAGE upregulation was clearly observed in these regions (Figure 1a,b). The number of RAGE/GFAPdouble immunopositive cells was also analyzed in the penumbra because astrocytes could be activated mainly in this area after an ischemic challenge [21,22]. RAGE was upregulated on all determined cell types (Figure 1c,d). Among cell types determined, it was highly expressed on Iba1-positive cells compared to that on cells expressing other brain cells  (Figure 1c,d). These results indicate that RAGE upregulation could occur mainly in activated microglia/macrophages of injured brains after an ischemic stroke. and CD31 (endothelial cell). The number of RAGE/Iba1-, RAGE/NeuN-, and RAGE/CD31-double immunopositive cells was analyzed in the ischemic core regions because RAGE upregulation was clearly observed in these regions (Figure 1a,b). The number of RAGE/GFAP-double immunopositive cells was also analyzed in the penumbra because astrocytes could be activated mainly in this area after an ischemic challenge [21,22]. RAGE was upregulated on all determined cell types (Figure 1c,d). Among cell types determined, it was highly expressed on Iba1-positive cells compared to that on cells expressing other brain cells markers (Figure 1c,d). These results indicate that RAGE upregulation could occur mainly in activated microglia/macrophages of injured brains after an ischemic stroke.

Suppressing LPA5 Activity Attenuates RAGE Upregulation in Injured Brains after tMCAO Challenge
Given data that RAGE could be upregulated in injured brains after tMCAO challenge (Figure 1), we determined whether LPA5 could be associated with such RAGE upregulation. To address this, we employed TCLPA5, an LPA5 antagonist, which could attenuate

Suppressing LPA 5 Activity Attenuates RAGE Upregulation in Injured Brains after tMCAO Challenge
Given data that RAGE could be upregulated in injured brains after tMCAO challenge (Figure 1), we determined whether LPA 5 could be associated with such RAGE upregulation. To address this, we employed TCLPA5, an LPA 5 antagonist, which could attenuate brain injuries after tMCAO challenge [9,10]. We chose 3 days after tMCAO challenge as the time point for analysis because RAGE upregulation was clearly observed at this time point (Figure 1a,b). We first determined expression levels of RAGE mRNA by qRT-PCR analysis. RAGE mRNA expression levels were significantly increased in the vehicle-administered group compared to those in the sham group (Figure 2a). TCLPA5 administration significantly attenuated such increase in injured brains (Figure 2a). Next, we determined expression levels of RAGE protein at 3 days after tMCAO challenge by Western blot analysis. The protein expression levels of RAGE were significantly upregulated in injured brains (Figure 2b,c). However, TCLPA5 administration significantly attenuated such RAGE upregulation (Figure 2b,c). Attenuated RAGE upregulation by TCLPA5 administration was reaffirmed by immunohistochemical analysis. The number of RAGE-positive cells was significantly increased in the penumbra and ischemic core regions of injured brains at 3 days after tMCAO challenge (Figure 2d-f). However, TCLPA5 administration signif-  (Figure 2d-f). These results indicate that LPA 5 could regulate RAGE upregulation in injured brains after an ischemic stroke. injured brains (Figure 2b,c). However, TCLPA5 administration significantly attenuat such RAGE upregulation (Figure 2b,c). Attenuated RAGE upregulation by TCLPA5 a ministration was reaffirmed by immunohistochemical analysis. The number of RAG positive cells was significantly increased in the penumbra and ischemic core regions injured brains at 3 days after tMCAO challenge (Figure 2d-f). However, TCLPA5 adm istration significantly reduced the number of RAGE-positive cells in both regions (Figu 2d-f). These results indicate that LPA5 could regulate RAGE upregulation in injur brains after an ischemic stroke. (d-f) Expression levels of RAGE protein in the penumbra and ischemic core regions at 3 days after tMCAO challenge were determined by immunohistochemica analysis. Representative images of RAGE-immunopositive cells in each region (d) and quantifica tion of their numbers ((e), the penumbra region; (f), the ischemic core region) are shown. Scale b 50 µ m. n = 5 mice per group. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus sham; # p < 0.05, ## p< 0.01, and ### p < 0.001 versus vehicle-administered tMCAO group.
In the current study, we demonstrated that RAGE upregulation could occur main in activated microglia/macrophages of injured brains after tMCAO challenge (Figu  1c,d). Therefore, we next determined whether suppressing LPA5 activity could attenua RAGE upregulation on activated microglia/macrophages by RAGE/Iba1 double immun fluorescence analysis. The number of RAGE/Iba1-double immunopositive cells was s nificantly increased in the ischemic core regions at 3 days after tMCAO challenge (Figu  3a,b). However, TCLPA5 administration significantly reduced the number of RAGE/Iba Representative images of RAGE-immunopositive cells in each region (d) and quantification of their numbers ((e), the penumbra region; (f), the ischemic core region) are shown. Scale bar, 50 µm. n = 5 mice per group. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus sham; # p < 0.05, ## p< 0.01, and ### p < 0.001 versus vehicle-administered tMCAO group.
In the current study, we demonstrated that RAGE upregulation could occur mainly in activated microglia/macrophages of injured brains after tMCAO challenge (Figure 1c,d). Therefore, we next determined whether suppressing LPA 5 activity could attenuate RAGE upregulation on activated microglia/macrophages by RAGE/Iba1 double immunofluorescence analysis. The number of RAGE/Iba1-double immunopositive cells was significantly increased in the ischemic core regions at 3 days after tMCAO challenge (Figure 3a,b). However, TCLPA5 administration significantly reduced the number of RAGE/Iba1-double immunopositive cells (Figure 3a,b). These results indicate that LPA 5 could upregulate expression of RAGE on activated microglia/macrophages in injured brains after an ischemic stroke.

Suppressing LPA 5 Activity Attenuates BBB Disruption in Injured Brains after tMCAO Challenge
RAGE can contribute to BBB disruption in CNS diseases including brain ischemic stroke [15,16,23]. Therefore, we addressed roles of LPA 5 in BBB disruption after ischemic stroke. For this, we determined whether suppressing LPA 5 activity by TCLPA5 administration could attenuate BBB disruption in injured brains at 3 days after tMCAO challenge through claudin-5/CD31 double immunofluorescence staining. The number of Life 2021, 11, 80 7 of 12 claudin-5/CD31-double immunopositive cells in injured brains after tMCAO challenge was markedly decreased compared to that in the sham group (Figure 4a,b), whereas TCLPA5 administration significantly increased it (Figure 4a,b). These data indicate that LPA 5 could contribute to BBB disruption in injured brains after an ischemic stroke.
Life 2021, 11, x FOR PEER REVIEW 7 of double immunopositive cells (Figure 3a,b). These results indicate that LPA5 could upreg ulate expression of RAGE on activated microglia/macrophages in injured brains after a ischemic stroke.

Suppressing LPA5 Activity Attenuates BBB Disruption in Injured Brains after tMCAO Challenge
RAGE can contribute to BBB disruption in CNS diseases including brain ischem stroke [15,16,23]. Therefore, we addressed roles of LPA5 in BBB disruption after ischem stroke. For this, we determined whether suppressing LPA5 activity by TCLPA5 admin istration could attenuate BBB disruption in injured brains at 3 days after tMCAO challeng through claudin-5/CD31 double immunofluorescence staining. The number of claudin 5/CD31-double immunopositive cells in injured brains after tMCAO challenge was mark edly decreased compared to that in the sham group (Figure 4a,b), whereas TCLPA5 ad ministration significantly increased it (Figure 4a,b). These data indicate that LPA5 coul contribute to BBB disruption in injured brains after an ischemic stroke.

LPA 5 Mediates Activation of ERK1/2 and NF-κB in Injured Brains after tMCAO Challenge
ERK1/2 and NF-κB are known to be involved in RAGE-dependent BBB disruption [16,19]. Therefore, the role of LPA 5 in activation of these signaling molecules in injured brains after ischemic challenge was addressed. First, we determined whether suppressing LPA 5 activity could attenuate ERK1/2 activation in injured brains at 3 days after tMCAO challenge by Western blot analysis. ERK1/2 phosphorylation was significantly increased in injured brains after tMCAO challenge compared to that in the sham group (Figure 5a,b). When LPA 5 activity was suppressed by TCLPA5 administration, such ERK1/2 phosphorylation was significantly attenuated (Figure 5a,b). Activated microglia/macrophages are main loci for NF-κB activation in post-ischemic brains [8,24]. The current study clearly showed RAGE upregulation on activated microglia/macrophages after tMCAO challenge (Figure 1c,d). Thus, we next determined whether suppressing LPA 5 activity could attenuate NF-κB activation in activated microglia/macrophages of injured brains by NF-κB/Iba1 double immunofluorescence staining. The number of NF-κB/Iba1double immunopositive cells was significantly increased in injured brains at 3 days after tMCAO challenge compared to that in the sham group (Figure 5c,d). However, suppressing LPA 5 activity by TCLPA5 administration significantly reduced their number in injured brains (Figure 5c,d). These results indicate that LPA 5 could regulate activation of ERK1/2 and NF-κB in injured brains after an ischemic stroke.

Discussion
LPA5 can contribute to acute brain injuries along with neuroinflammatory responses in injured brains after ischemic stroke, including activation of microglia/macrophages and

Discussion
LPA 5 can contribute to acute brain injuries along with neuroinflammatory responses in injured brains after ischemic stroke, including activation of microglia/macrophages and production of proinflammatory cytokines [9]. Suppressing LPA 5 activity with its antagonist, TCLPA5, can attenuate such acute brain injuries and neuroinflammatory responses in mice after ischemic challenge [9]. In addition, it can attenuate chronic brain injuries in mice after ischemic challenge by enhancing neurogenesis and angiogenesis [10]. These two independent studies clearly suggest that LPA 5 can play pathogenic roles in brain injuries during both acute and chronic phases after ischemic stroke. Results of the current study suggest that RAGE is a pathogenic factor for LPA 5 -mediated brain injuries after ischemic stroke. RAGE was upregulated during the acute phase after ischemic challenge (at 1 day and 3 days after tMCAO challenge) in the penumbra and ischemic core regions, particularly on activated microglia/macrophages, whereas suppressing LPA 5 activity with TCLPA5 attenuated such a RAGE upregulation. Suppressing LPA 5 activity with TCLPA5 also attenuated RAGE-relevant BBB disruption in injured brains after tMCAO challenge. As underlying signaling pathways, LPA 5 could be associated with activation of ERK1/2 and NF-κB, both of which are effector pathways of RAGE [25,26]. They might be involved in BBB disruption [27]. All of these data clearly suggest that LPA 5 could regulate the pathogenesis of brain ischemic stroke via RAGE, at least in part.
It is well-known that RAGE plays pathogenic roles in various diseases [11,23], including brain ischemic stroke [14][15][16]. Suppressing RAGE activity by either a pharmacological antagonist or genetic deletion can attenuate ischemic brain injuries including brain infarction, neurological functional deficit, and neuronal survival in different rodent models such as permanent MCAO (pMCAO)-challenged mice [14], intracerebral hemorrhage (ICH)challenged rats [16], and bilateral common carotid artery occlusion (BCCAO)-challenged mice [15]. Such a pathogenic role of RAGE is supported by its upregulation at both mRNA and protein levels in injured brains of ischemic stroke-challenged rodents [15,16,20] and human stroke patients [20]. In addition, immunohistochemical analyses have suggested regional and cell type-specific RAGE upregulation after ischemic challenge [15,16,20]. The penumbra region has been demonstrated to be the area that RAGE is upregulated after an ischemic challenge in pMCAO-challenged mice [20]. In view of cell types, endothelial cells, activated microglia/macrophages, and neurons have been suggested as cell types where RAGE can be upregulated after ICH or BCCAO challenge [15,16]. In the current study using tMCAO-challenged mice, RAGE was upregulated in post-ischemic brains at 1 day and 3 days after tMCAO challenge, with higher expression levels at 3 days after tMCAO challenge than at 1 day after such challenge. Interestingly, RAGE upregulation was observed in the penumbra and ischemic core regions, but not in periischemic regions, with a stronger immunoreactivity for RAGE in the ischemic core region than in the penumbra region. Moreover, the current study revealed that RAGE upregulation occurred mainly in activated microglia/macrophages of injured brains after tMCAO challenge. This could be supported by previous studies showing that RAGE is upregulated on activated microglia/macrophages in injured brains after ICH challenge [16] or on cultured microglia subjected to oxygen and glucose deprivation as an in vitro ischemic challenge [14]. In addition to activated microglia/macrophages, the current study demonstrated that RAGE could be upregulated on neurons and endothelial cells of injured brains after tMCAO challenge as demonstrated in previous studies using other stroke models [15,16]. All these independent studies including ours strongly indicate that RAGE can be upregulated in injured brains after ischemic challenge, leading to ischemic brain injuries. It is of note that LPA 5 can lead to RAGE upregulation in post-ischemic brains. Suppressing LPA 5 activity with TCLPA5 administration attenuated RAGE upregulation at mRNA and protein levels in injured brains after tMCAO challenge. Moreover, it attenuated RAGE upregulation in both the penumbra and ischemic core regions, particularly on activated microglia/macrophages, after tMCAO challenge. Considering that our previous studies have shown that LPA 5 could contribute to tMCAO-induced brain damages [9], the current study indicates that RAGE can be involved in LPA 5 -mediated ischemic brain injuries as an underlying pathogenic mediator. Interestingly, LPA 5 can be upregulated in the ischemic core regions, particularly on activated microglia/macrophages, but not on neurons [9]. Therefore, it might be possible that LPA 5 regulate RAGE expression in activated microglia/macrophages after ischemic challenge.
RAGE is known to regulate inflammatory responses through activation of ERK1/2 and NF-κB [25,26], both of which are associated with BBB disruption [27], indicating that RAGE can contribute to BBB disruption through activation of these signaling molecules. In fact, suppressing RAGE activity with FPS-ZM1, a RAGE-specific antagonist, can significantly attenuate BBB dysfunction in radiation-induced endothelial barrier injury through inactivation of ERK1/2 and NF-κB [19]. It can also attenuate BBB disruption in injured brains after ICH challenge, along with downregulation of NF-κB and proinflammatory mediators such as interleukins, inducible nitric oxide synthase, cyclooxygenase-2, and matrix metalloproteinase-9 [16]. Similarly, the current study demonstrated that suppressing LPA 5 activity with TCLPA5 administration could attenuate BBB disruption, ERK1/2 activation, and NF-κB activation in injured brains after tMCAO challenge, indicating that suppressing RAGE expression by inhibiting LPA 5 activity might lead to such attenuated events. The current study also demonstrated that NF-κB activation and RAGE upregulation occurred in activated microglia/macrophages after tMCAO challenge whereas TCLPA5 administration attenuated both events. In fact, activated microglia/macrophages can contribute to BBB disruption in many brain diseases, including brain ischemic stroke [28,29]. Moreover, LPA 5 can contribute to the activation of microglia/macrophages and subsequent neuroinflammatory responses, such as upregulation of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in post-ischemic brains [9]. These proinflammatory cytokines have been considered as contributing factors for BBB disruption after ischemic challenge [30,31]. Therefore, RAGE might play important roles in LPA 5 -mediated BBB disruption and neuroinflammatory responses in post-ischemic brains possibly by regulating the activation of ERK1/2 and NF-κB.

Conclusions
The current study demonstrates that RAGE can be involved in LPA 5 -mediated brain injury following ischemic stroke, along with experimental evidence for roles of LPA 5 in RAGE-relevant BBB disruption and signaling pathways, such as ERK1/2 and NF-κB in post-ischemic brains. LPA 5 has been suggested as a pathogenic mediator for other disease types, such as neuropathic pain [32,33], psoriasis [34], demyelination [35], and itching [36]. Findings of the current study on a possible involvement of RAGE in LPA 5 -mediated pathogenesis in ischemic stroke might add clues for understanding how LPA 5 can play its pathogenic roles in such diseases. In addition, targeting LPA 5 might be a possible strategy to treat various diseases involving RAGE as a pathogenic factor.