
Selective Epac2 antagonist attenuates cerebral infarction induced by secondary brain injury in rats
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- Published online on: July 10, 2025 https://doi.org/10.3892/etm.2025.12922
- Article Number: 172
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Copyright: © Kakei et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Cerebrovascular events, particularly acute cerebral infarction, rank as the second most common cause of mortality and the third most common cause of disability on a global scale. Acute cerebral infarction represents the predominant subtype of stroke. The key element in managing cerebral infarction is the timely restoration of blood flow in the blocked artery to salvage the ischemic penumbra (1,2). Current research indicates that mechanical thrombectomy is advantageous for individuals with large vessel blockage occurring within 6 h of the onset of a stroke (3-6), and successful reperfusion is achieved in up to 90% of patients (7). However, analyses of retrospective metadata suggest that over 50% of these individuals do not exhibit clinical enhancements, with some experiencing deteriorating symptoms, typically linked to primary brain injury (PBI) and secondary brain injury (SBI) in the affected brain regions (8,9).
PBI related to ischemia typically occurs due to direct or indirect harm that causes a disruption in cellular energy. On the other hand, SBI is the outcome of a series of processes triggered by PBI and its associated metabolites (10). The series of events in SBI encompasses all subsequent pathophysiological alterations following a cerebral infarction, including adverse consequences resulting from glutamate excitotoxicity, stimulation of oxidative stress and inflammatory responses (11), stimulation of apoptosis (12), and blood-brain barrier (BBB) disruption leading to the entry of complement proteins, causing vasogenic and cytotoxic edema (13).
Exchange protein directly activated by cyclic adenosine monophosphate (cAMP) (Epac) was initially discovered in 1998 and has subsequently been associated with the development of chronic obstructive pulmonary disease and cardio-vascular disease (14). Initially, cAMP was considered to function by stimulating cAMP-dependent protein kinase A (PKA) or engaging with cyclic nucleotide binding ion channels. PKA transmits cAMP signals through the direct phosphorylation of target proteins, whereas Epac-mediated signaling primarily relies on the activation of the small guanosine triphosphatases Rap, Rap1 and Rap2 (15,16). Epac2 is implicated in apoptosis after intracerebral hemorrhage and head trauma (17,18), particularly the p38-mitogen-activated protein kinases (MAPKs), and cellular proliferation and differentiation (19). However, the potential contribution of Epac in the development of SBI or apoptosis pathways following cerebral infarction remains unexplored. This research aimed to investigate, 1) The mechanisms by which Epac2, p38 pathway mediates apoptosis in the brain following cerebral infarction, 2) The proposition that the selective inhibition of Epac2 using 1,3,5-trimethyl-2-[(4-methylphenyl)sulfonyl]-benzene (ESI-05) can effectively inhibit MAPKs pathways and inflammatory cell infiltration, while also decreasing brain edema in a rat model of permanent middle cerebral artery (MCA) occlusion.
Materials and methods
Ethics
All experimental procedures were approved by the Nihon University Animal Experiment Committee (permission number: AP21DEN024-1 and AP21MED020-1), and were conducted in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and adhered to the ARRIVE guidelines.
Animals and experimental groups
A total of 31 male Sprague-Dawley rats (9 to 11 weeks old, 290 to 330 g) were obtained from Oriental Yeast Co., Ltd. The animals were maintained in accordance with typical animal housing standards, with environmental conditions set at a temperature of 23±1˚C, humidity of 50-60%, and a light-dark cycle of 12 h each, while being provided ad libitum access to food and water.
Briefly, rats were initially induced with anesthesia using a combination of 5% isoflurane, 65% nitrous oxide, and 30% O2, followed by maintenance anesthesia with 1 to 2% isoflurane, 68 to 69% nitrous oxide, and 30% O2 administered through a face mask. During the surgical procedure, a temperature probe was placed into the rectum to monitor and regulate the body temperature at 37˚C using a temperature control pad.
Permanent MCA occlusion was achieved through intraluminal vascular occlusion using a silicone-coated filament and a 4-0 siliconized suture (Doccol Corp., Sharon, MA) following established protocols (20). The micro-tip was positioned at a right angle to the right parietal skull surface to observe blood circulation in the MCA region using laser Doppler flowmetry. Placement was confirmed by the slight resistance encountered at approximately 19 mm from the arteriotomy incision. Additionally, a simultaneous decrease in cerebral blood flow measurement by less than 30% compared to the initial level was confirmed using laser Doppler flowmetry as evidence of ischemia.
The rats were allocated into three distinct groups through a random process: the sham group (n=9) which underwent solely ligation of the internal carotid artery, the permanent MCA occlusion-control group (n=11) treated with 30% dimethyl sulfoxide (DMSO) (8 ml) post ischemia, and the permanent MCA occlusion-ESI-05 group (n=11) administered with a specific Epac2 inhibitor (8 mg/kg dissolved in 30% DMSO, intraperitoneally) following the ischemic event. ESI-05 (SML1907; Sigma-Aldrich Japan, Tokyo, Japan) was solubilized in DMSO (043-07216; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), with DMSO serving as the control substance in this study. After the surgical procedure, the rats were transferred to a heated recovery chamber until they regained ambulatory function, after which they were housed in separate cages. Neurological function was assessed using the modified Garcia score. Score were determined shortly at 1 and 24 h after onset of ischemia (sham: no data, control group: n=11 and ESI-05 group: n=11).
Humane endpoints
In this study, rats that lost >10% of their preoperative body weight after surgery, displayed a lack of activity in terms of exercise, eating, or drinking, or exhibited signs of infection were classified as hyper-invasive and were humanely euthanized. It is noteworthy that no rats met these criteria in the present investigation.
Evaluation of stroke volume and brain edema
At the 24-h mark, all remaining animals were euthanized in order to assess stroke volume and brain edema. The animals were initially sedated using 5% isoflurane and then maintained under anesthesia with 2 to 3% isoflurane. Subsequently, they were perfused transcardially with cold physiological saline until the outflow from the right atrium appeared clear. The brain was meticulously extracted and divided into six 2-mm coronal segments, commencing from the junction of the olfactory bulb and frontal lobe. Sections No. 1-6 were prepared in this manner. Sections No. 3 and 4 were submerged in a 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate buffered saline (PBS) for a duration of 15 min at a temperature of 37˚C (21). Sections No. 5 and 6 were preserved as samples for western blotting (sham: n=9, control group: n=9 and ESI-05 group: n=9).
After TTC staining, each brain slice was gently dried with water absorbent paper to eliminate any remaining excess liquid and then photographed alongside a reference ruler. The determination of swelling volume was conducted in accordance with established methodologies as described in previous literature (22-24). Specifically, four distinct regions on each brain slice were measured individually using ImageJ software (v1.48, National Institutes of Health). These regions included the contralateral hemisphere volume, total ipsilateral hemisphere volume, ipsilateral TTC-positive volume, and TTC-negative volume. The contralateral hemisphere volume represented the baseline hemisphere volume. Subsequently, the calculation of the ‘indirect’ stroke volume was performed.
Brain water content was assessed through the modified wet-dry weight technique as outlined in a previous study (25). Initially, the wet weight of the sections No. 3 and 4 were recorded, followed by subjecting the sample to heating in an oven at 100˚C for a duration of 24 h to obtain the dry weight measurement.
Western blotting
The supernatant sample was diluted 20-fold, and the concentration of total protein was determined using the RC DC Protein assay Kit (5000122JA; Bio-Rad Laboratories, Inc., Hercules, CA). A spectrophotometer (Model 680; Bio-Rad Laboratories, Inc.) was employed for quantifying the concentration. The samples underwent lysis through the utilization of Laemmli sample buffer (1610737; Bio-Rad Laboratories, Inc.) and beta-mercaptoethanol (1610710; Bio-Rad Laboratories, Inc.). A 15-µg aliquot was loaded per well as the total protein content. The separation of proteins was carried out through polyacrylamide gel electrophoresis, with electrophoresis conducted at 120 V and 400 mA for a duration of 65 min using a gradient gel with a concentration range of 4 to 20% (catalog number 567-1094; Bio-Rad Laboratories, Inc.). The Precision Plus Protein Two Color Standard (catalog number 1610374; Bio-Rad Laboratories, Inc.) served as the molecular weight reference marker. The iBlot system (catalog number IB401001; Thermo Fisher Scientific Inc., Waltham, MA) was employed for the transfer process.
In the experimental procedure, the primary antibody response involved the utilization of anti-Epac2 antibody (ASC11419; Abcepta, San Diego, CA) at a 1,000-fold dilution, anti-cleaved caspase 3 antibody (Asp175; Cell Signaling Technology, Danvers, MA) at a 1,000-fold dilution, phospho-p38 antibody (ab38238; Abcam, Cambridge, UK) at a 500-fold dilution, and anti-beta-actin antibody (GTX109639; GeneTex, Inc., Irvine, CA) at a 20,000-fold dilution, serving as an internal control. The process of antibody sensitization and anti-beta-actin antibody sensitization was conducted at a temperature of 4˚C for a duration of 24 h. A secondary antibody reaction was carried out using anti-rabbit immunoglobulin G antibody (AP182P; Millipore Sigma, Burlington, MA) and sensitized at room temperature for a period of 1 h.
Bands were identified utilizing a ChemiDocXRS detector (Bio-Rad Laboratories, Inc.) in conjunction with the enhanced chemiluminescence technique (WP20005; Thermo Fisher Scientific Inc.) for visualization. Measurement analysis was conducted using Image Lab software (version 6.0; Bio-Rad Laboratories, Inc.). Control samples with known concentrations were added to all gels to standardize staining intensity across different gel runs. The control samples were derived from the same brain tissue and were diluted to 25, 50, and 75% concentrations for this study. The intensity of the band was quantified relative to the positive control and expressed as a ratio. The findings were then analyzed in terms of the ratio of the target protein to beta-actin. The activation level of the target protein was assessed by calculating the ratio of the phosphorylated (activated) form to the total protein.
Immunohistochemical staining
Immunostaining was performed to evaluate the expression of Epac2, Cleaved caspase 3, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) and Fluoro Jade C (FJC) staining 24 h after ischemia (sham: no data, control group: n=2 and ESI-05 group: n=2). Samples were perfused and preserved using paraformaldehyde. The brain that had been frozen was cut into sections that were 20-µm thick, with intervals of 500-µm, using a cryostat. Tissues were suspended in PBS solution for immunostaining. Initially, the tissue sections were subjected to a 3% hydrogen peroxide solution to eliminate the endogenous peroxidase activity. Subsequently, the sections were blocked using 2% horse serum, followed by incubation with the primary antibody at 4˚C overnight. The VECTASTAIN Elite ABC HRP Kit (PK-6101, 6102; Vector Laboratories, Burlingame, CA) was employed for the secondary antibody reaction through the diaminobenzidine development process. Following this, the section underwent dehydration using alcohol and xylene, was cleared, and subsequently covered.
TUNEL staining
The TUNEL assay was used to analyze apoptotic cell death. The tissue samples underwent a process of removal of wax and dehydration by exposure to heat at 70˚C for 60-90 min in an oven, followed by rehydration using a series of xylene and ethanol-water gradients. Subsequently, the samples were treated with Triton X-100 for a duration of 10 min. After washing three times with PBS, the tissue sections were exposed to the TUNEL reaction mixture from the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Nippon Chemi-Con Corporation, Tokyo, Japan) at 37˚C for 1 h. Subsequently, the sections were washed three more times with PBS and then treated with a fade-resistant blocking medium that included the diaminobenzidine substrate kit (ab64238; Abcam). After staining, the tissues were transferred onto a slide glass that had been coated, dehydrated, and covered with a slip.
FJC staining
FJC staining was used for detection of damaged neurons. The tissue slices were rehydrated using water and permeated with a solution containing 0.04% Triton X-100. Subsequently, the sections were treated with the FJC Ready-to-Dilute Staining Kit (TR-100-FJT; Funakoshi, Tokyo, Japan). The sections were stained with 4',6-diamidino-2-phenylindole and analyzed by laser confocal microscopy.
Neurological deficit evaluation
Neurological function was evaluated utilizing the modified Garcia score as outlined in a previous study (26). The scores were recorded within 24 h following the onset of ischemia. In cases where animals did not survive, a score of 0 was allocated.
Statistical analysis
Statistical analysis was conducted utilizing the SPSS Statistics Software (version 21; IBM, Armonk, NY). Data were expressed as the mean ± standard deviation. Statistical analysis was performed using an unpaired t-test for the comparison of two groups and a one-way ANOVA for the comparison of three groups (western blotting data). The data was analyzed using one-way ANOVA, with adjustments made to the degrees of freedom using epsilon for datasets exhibiting unequal variances. Additional tests were conducted only in cases where the analysis of variance revealed significant differences among the factors. Subsequent testing was carried out using the Tukey method. Data were expressed as the median and interquartile range (IQR), and statistical analysis for the comparison of two groups was performed using the Mann-Whitney U-test for the neurological score, as this was an ordinal variable. All statistical tests were two-sided, and a P-value less than 0.05 was deemed to be statistically significant.
Data availability
All procedures and information will be provided upon request and in compliance with the data transfer regulations of the institution.
Results
Stroke volume, brain edema, and neurological outcome
Indirect stroke volume in all animals was 70.3±5.1% of the ipsilateral hemisphere. ESI-05 administration significantly reduced infarct volume (Fig. 1A and B). Comparison of treatment arms showed ESI-05 administration reduced indirect stroke volume relative to the control group (Fig. 1C, 65.3±2.5% vs. 72.9±2.1%, P<0.05).
Swelling volume of the ischemic hemisphere was decreased by ESI-05 treatment. This reduction in swelling volume corresponded with a decrease in the estimated water content of the ischemic hemisphere compared to the control group (Fig. 1D, 81.7±1.2% vs. 82.5±0.6%, P<0.05).
Neurological function following ischemia showed no significant difference between the two groups, as indicated by comparable scores (ESI-05 group: median, 11 points; IQR, 10-11 points; vs. Control group: median, 11 points; IQR, 10.5-11 points; P=0.68). Animals treated with ESI-05 had higher average score relative to the control group (Fig. 1E; median, 14 points; IQR, 13.5-14 points; vs. median, 10 points; IQR, 9.0-11 points; P<0.001) after 24 h, consistent with improvement in neurological function (Table I).
![]() | Table INeurological score comparison between the control and ESI-05 groups at 1 and 24 h post-surgery. |
Epac2 expression after ischemia
Epac2 expression was evaluated in brain tissue removed at 24 h after permanent MCA occlusion. Staining with Epac2 antibody was observed in sections from the control group (Fig. 2A). Cells expressing the positive marker were observed throughout the ischemic hemisphere, encompassing the peri-infarct region. Conversely, a reduced number of positive cells were detected in the ESI-05 treatment group, as illustrated in Fig. 2B.
Regarding the number of samples indicated in the Materials and Methods, some western blot samples were missing or showed poor detection of the target or loading control proteins. Therefore, the data were analyzed using the following sample numbers: Sham: n=8, Control: n=8, ESI-05: n=6.
Western blotting was employed to investigate the Epac2 expression levels. Values relative to beta-actin were calculated for the target protein. Epac2 expression was notably elevated in the peri-infarct area in both the control group (P<0.001) and ESI-05 group (P<0.05) compared to the sham group. Epac2 expression was notably reduced in the ESI-05 group compared to the control group (P<0.05) [F (2, 19)=18.9, P<0.001] (Figs. 2C and 3C).
Inhibition of Epac2 attenuated p38-MAPK signaling pathway after permanent MCA occlusion
Immunofluorescence staining and western blotting were employed to investigate the role of Epac2 in neural cell death by examining caspase 3, phospho-p38, and TUNEL staining. Results from both immunofluorescence staining and western blotting indicated a notable reduction in caspase-3 expression within the ESI-05 group [F (2, 19)=31.3, P<0.001] (Fig. 3A-C and F; P<0.01, P<0.001).
The levels of phosphorylated p38 in the peri-infarct region showed a notable increase in both the control and ESI-05 treatment groups compared to the sham group. However, no statistically significant variance was observed between the control and ESI-05 groups (P>0.05). In contrast, the levels of phosphorylated p38 significantly rose following the permanent MCA occlusion, which was subsequently counteracted by the administration of ESI-05 [F (2, 19)=35.7, P<0.001] (Fig. 3C, E and G; P<0.05, P<0.001).
TUNEL staining revealed that suppression of Epac2 effectively mitigated the escalation of apoptotic cell death. Additionally, FJC staining indicated that inhibition of Epac2 substantially hindered the degeneration of neurons (Fig. 4).
These findings suggested that suppressing Epac2 could potentially mitigate neural cell apoptosis triggered by cerebral ischemia, with a potential mechanism involving the suppression of p38-MAPK signaling pathway phosphorylation.
Discussion
This study investigated the molecular function of Epac2 in the occurrence of secondary brain damage following ischemic stroke. Treatment with the Epac2-specific inhibitor, ESI-05, reduced neuronal cell death in the peri-infarct region of the brain. Control of Epac2 activity decreased phosphorylation of p38 and activation of caspase 3, resulting in reduced neuronal cell death and, consequently, alleviation of brain edema.
cAMP, a second messenger molecule, activates guanine nucleotide-binding proteins, primarily through PKA, and functions as a regulatory factor in various cellular processes. However, cAMP was found to stimulate insulin secretion in pancreatic beta-cells even in the presence of PKA inhibition (14,27). The protein mediating this action is collectively referred to as Epac. Epac exhibits distinct localization and actions, and acts as an intermediate in major cAMP-dependent signaling pathways implicated in conditions such as tumors, heart failure, diabetes, inflammatory diseases, infections, and neurological disorders (28). The cAMP/PKA pathway has been reported to suppress neuronal cell death associated with stroke by maintaining the generation of free radicals and intracellular Ca ion regulation (29). PKA activation has been associated with neuroprotection, leading to reduced stroke volume. However, the interactions between stroke, Epac2, and PKA signaling regulation remain unclear.
Epac1 is activated by cAMP, then interacts with Ras proteins to promote cell adhesion through integrins (30). Many cytokines and growth factors are regulated by the activation of Ras (31). Epac activation has been reported to inhibit macrophage cytoskeletal reorganization and phagocytosis (32). Inhibition of Epac-Rap1 signaling reduces mitochondrial reactive oxygen species production, so increasing the risk of arrhythmias due to early depolarization in cardiac muscle (33).
Epac2 is involved in the calcium ion signaling of pancreatic beta-cells to stimulate insulin secretion (34). Epac2 promotes intracellular calcium ion release, activates endoplasmic reticulum adenosine triphosphatase, and enhances mitochondrial dehydrogenase activity, leading to increased cytoplasmic adenosine triphosphate (ATP) levels. Consequently, the ATP sensitivity of K channels is increased which promotes insulin secretion. Additionally, Epac2 is known to close ATP-sensitive K channels on the cell membrane, with resultant membrane depolarization (34). The present study revealed that Epac2 expression is up-regulated after stroke.
Brain edema is a severe complication of ischemic stroke, associated with deteriorated function and worsened prognosis (35). The occurrence of brain edema is crucial in the progression of ischemic stroke damage. Even a slight increase in brain water content leads to a significant rise in intracranial pressure with resultant deterioration in outcomes (36). Therefore, reducing brain edema is considered a promising therapeutic approach for managing intracranial pressure after ischemic stroke. This study confirmed that controlling Epac2 activity could mitigate increases in brain water content in the infarct hemisphere after stroke.
Previous studies have suggested a relationship between Epac2 and cell death in myocardial ischemia. Activation of Epac2 and extracellular signal-regulated kinases 1/2 is involved in cell survival by reducing cell necrosis (37). Epac2 inhibition in lung cancer cells decreases cisplatin-induced apoptosis through the Epac2-Rap1-Akt pathway (38). Epac2 activation also inhibits cAMP-dependent cell differentiation and proliferation in neuroendocrine cells by activating Rap2A (39). In the present study, immunostaining confirmed the expression of Epac2 in the peri-infarct region after stroke.
cAMP and Epac actively participate in neuronal functions such as signal transduction and neural plasticity induced by neurotransmitters (32). Epac expression in cardiac muscle cells stimulates cAMP signaling to mitigate neuronal cell death under various stress conditions (40). Administration of the selective Epac2 inhibitor ESI-05 helps to improve neurological function in animal models of brain hemorrhage and traumatic brain injury (17,18). In this study, ESI-05 administration was observed to suppress Epac2 expression, consistent with the western blotting results.
p38-MAPK, activated by phosphorylation, is crucial in regulating inflammation, apoptosis, autophagy, cell survival, and cell death (18,41,42). Enhanced p38-MAPK signaling promotes neuronal cell death, whereas attenuation has neuroprotective effects (39). Epac2 is involved in differentiation through the p38-MAPK signaling pathway, suggesting that Epac2 expression may induce cell growth arrest (39). Epac2 is also involved in nociception through the p38 pathway in a rat model of plantar incision (43). The present study suggests that post-stroke Epac2 may induce apoptosis through the p38-MAPK signaling pathway and confirmed that controlling Epac2 could weaken the p38-MAPK signaling pathway.
Caspase 3, a cysteine protease, is crucial in apoptosis and is influenced by the mitochondrial apoptosis pathway, so is essential in cell apoptosis (44). Caspase 3 has a central function in neuronal cell apoptosis after stroke (45). Stimulation of the caspase 3 pathway is involved in chromatin condensation and DNA fragmentation in apoptosis. DNA fragmentation is suppressed in cells undergoing apoptosis in caspase 3-deficient cell lines (46). Additionally, the caspase 3 inhibitor z-DEVD-fmk significantly inhibits apoptosis in brain tissue cells in a rat stroke model, so demonstrating protective effects on brain tissue (45).
Previous research suggests that inhibiting the p38-MAPK pathway enhances the expression of occludin, a major tight junction protein of the BBB, thus maintaining its function (47). Inhibition of the p38-MAPK pathway also reduces the expression of aquaporin 4 associated with edema, so alleviating brain edema (47). In this study, suppression of Epac2 expression was associated with inhibition of p38 activation, so potentially reducing BBB permeability. Epac2 may affect the factors contributing to post-stroke brain edema. Further investigation is required to understand the involvement of Epac2 in the post-stroke inflammatory and immune responses.
In this study, increased Epac2 expression was associated with apoptosis after stroke. Moreover, the post-stroke increase in Epac2 activity was accompanied by a significant increase in cleaved caspase 3, indicating increased apoptosis in the brain. TUNEL staining further confirmed the increase in post-stroke neuronal cell death with the upregulation of Epac2 and cleaved caspase 3.
The present study has several limitations. The specific interactions between Epac2 and ERK, JNK-MAPK pathway have not been extensively detailed, but given the complexity of cellular signaling networks, indirect interactions could exist. The temporal changes of Epac2 after ischemic stroke remain unclear. Such temporal alterations, including both short and long-term changes, must be clarified, as well as the effective time window associated with various drug administration timings. Additionally, Epac2 is known to be involved in the signal transduction of pancreatic β-cells, so enhancing insulin secretion. Further research is needed to investigate the impact of ESI-05 administration post-stroke on blood glucose levels. Clarification of these effects may improve post-stroke functionality through the clinical control of Epac2. One of the limitations of this study is the lack of histological evaluation in the sham group. Although molecular biological assessments were conducted, the absence of histological analysis in the sham group may limit the comprehensive understanding of potential underlying cellular changes or tissue responses that could contribute to the observed outcomes. Finally, we mentioned the small sample size of immunohistochemical staining as a limitation of this study and it as an area for improvement in future research.
In conclusion, this research has shown that Epac2 plays a significant role in the development of ischemic stroke by activating the p38/caspase 3 pathway. Suppression of Epac2 expression leads to enhanced neurological function in a rat model of ischemic stroke. Epac2 is associated with neuronal cell apoptosis after ischemic stroke. Therefore, control of Epac2 may provide a novel therapeutic target for secondary brain damage after ischemic stroke.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by Japan Society for the Promotion of Science KAKENHI (grant nos. 21K09189 and 24K12273).
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
YK and TI designed all the experiments. YK, TI and RK performed the experiments. KS, HO, NO, MA and AY helped with data analysis and draft writing. YK, TI and AY reviewed the manuscript. YK, TI and AY confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.
Ethics approval and consent to participate
The present study was approved by the Animal Care and Use Committee at Nihon University School of Medicine (approval nos. AP21DEN024-1 and AP21MED020-1; Tokyo, Japan). All experiments were conducted according to the animal experimental protocol manuals at Nihon University School of Medicine. Efforts were made to avoid pain and distress to the animals.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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