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PLoS One
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10.1371/journal.pone.0306793
PONE-D-24-00772
Research Article
Medicine and Health Sciences
Neurology
Cerebral Ischemia
Medicine and Health Sciences
Medical Conditions
Cerebrovascular Diseases
Stroke
Ischemic Stroke
Medicine and Health Sciences
Neurology
Cerebrovascular Diseases
Stroke
Ischemic Stroke
Medicine and Health Sciences
Vascular Medicine
Stroke
Ischemic Stroke
Biology and Life Sciences
Anatomy
Nervous System
Central Nervous System
Blood-Brain Barrier
Medicine and Health Sciences
Anatomy
Nervous System
Central Nervous System
Blood-Brain Barrier
Biology and Life Sciences
Cell Biology
Cell Processes
Cell Death
Apoptosis
Biology and Life Sciences
Immunology
Immune Response
Inflammation
Medicine and Health Sciences
Immunology
Immune Response
Inflammation
Medicine and Health Sciences
Clinical Medicine
Signs and Symptoms
Inflammation
Medicine and Health Sciences
Neurology
Brain Damage
Biology and life sciences
Biochemistry
Nucleic acids
RNA
Non-coding RNA
Natural antisense transcripts
MicroRNAs
Biology and life sciences
Genetics
Gene expression
Gene regulation
MicroRNAs
Biology and life sciences
Cell biology
Signal transduction
Cell signaling
Signaling cascades
AKT signaling cascade
MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathway
miRNA-199a-5p attenuates BBB disruption following ischemic stroke by regulating PI3K/Akt signaling pathway
https://orcid.org/0000-0002-9080-5042
Ni Guangxiao Project administration 1 *
Kou Lulu Data curation Software 1
Duan Chunqiao Methodology Writing – original draft 1
Meng Ran Data curation Funding acquisition Methodology 1
Wang Pu Writing – original draft 2
1 Department of Rehabilitation of the Second Hospital of Hebei Medical University, Shijiazhuang, China
2 Stomatological Laboratory of the Second Hospital of Hebei Medical University, Shijiazhuang, China
Sessa Francesco Editor
University of Catania, ITALY
Competing Interests: All authors declare that they have no conflicts of interest to report.

* E-mail: ngx7912156@163.com
20 9 2024
2024
19 9 e03067931 2 2024
24 6 2024
© 2024 Ni et al
2024
Ni et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Objective

To explore whether miR-199a-5p regulated BBB integrity through PI3K/Akt pathway after ischemia stroke.

Methods

Adult male Sprague-Dawley rats with permanent middle cerebral artery occlusion(MCAO) were used in experiment. The Ludmila Belayev 12-point scoring was used to measure the neurological function of MCAO rats. The Evans Blue Stain, immunofluorescence staining, western-blotting and RT-PCR were performed to evaluate the effects of miR-199a-5p mimic on BBB integrity in rats following MCAO.

Results

The result suggested that miR-199a-5p mimic treatment possessed the potential to boost proprioception and motor activity of MCAO rats. MiR-199a-5p decreased the expression of PIK3R2 after MCAO, activated Akt signaling pathway, and increased the expression of Claudin-5 and VEGF in the ischemic penumbra. Furthermore, miR-199a-5p alleviated inflammation after cerebral ischemia. BBB leakage and neurocyte apoptosis were cut down in MCAO rats treated with miR-199a-5p mimic.

Conclusions

MiR-199a-5p mimic decreased the expression of PIK3R2 and activated Akt signaling pathway after ischemia stroke, reduced the expression of inflammatory cytokines, and attenuated BBB disruption after ischemic stroke.

http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81273609 https://orcid.org/0000-0002-9080-5042
Ni Guangxiao http://dx.doi.org/10.13039/501100003787 Natural Science Foundation of Hebei Province H2023206115 https://orcid.org/0000-0002-9080-5042
Ni Guangxiao National Natural Science Foundation of China, (No.81273609) Natural Science Foundation of Hebei Province, (No. H2023206115). Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files.
Data Availability

All relevant data are within the manuscript and its Supporting Information files.
==== Body
pmcIntroduction

Stroke has been the major risk threatening human health. China has become the region with the highest incidence rate and prevalence of stroke in the world. However, ischemic stroke accounts for approximately 60% -85% [1–3]. After stroke, disability severely reduced people’s quality of life [4,5]. following ischemic stroke, the expression of various inflammatory cytokines increased, leading to damage to the integrity of the blood-brain barrier (BBB) [6,7]. When cerebral ischemia occurred, the permeability of the BBB increased, harmful components in blood vessels seeped into brain tissue, exacerbating cerebral injury [5,8,9].

The disruption and dysfunction of the BBB leading to leukocyte infiltration, cerebral edema, and hemorrhagic transformation were common mechanisms [10]. Maintaining the integrity and normal function of the BBB to protect the central nervous system(CNS) from secondary damage was a potential therapeutic strategy for ischemic stroke [11]. The BBB was composed of endothelial cells, pericytes, astrocyte terminals, and basement membrane, regulated substance exchange between the CNS and peripheral blood circulation. The BBB played a key role in the homeostasis of the CNS [10,12]. Multiple factors were involved in regulating the structure and function of the BBB. Especially, the continuous tight junctions (TJs) between cells, which were the main structure ensured selective permeability of the BBB [10,13]. TJs were closed structures formed by the fusion of specific transmembrane proteins from adjacent cell membrane outer layers, located between brain microvascular endothelial cells [14]. TJs were mainly composed of various proteins such as transmembrane proteins [Claudin, Occludin], cytoplasmic attachment proteins, cytoskeletal proteins, and other proteins that regulated BBB permeability [15]. Particularly, claudin-5 was the predominant TJs protein that influenced the selective permeability of the BBB, and inflammation induced its downregulation and BBB disruption [11,15,16].

MicroRNA (miRNA) was a kind of endogenous non coding RNA with length about 19-25nt, which was widely involved in the post transcriptional regulation of genes. The mechanism of miRNA was to inhibit protein translation and induce target RNA degradation through incomplete miRNA mRNA complementary pairing. miRNA has become potential clinical biomarker for diagnosis and prognosis of many diseases [17–20]. MiR-199a-5p was widely expressed in brain tissues, such as the hippocampus, olfactory bulb, subventricular zone, cortex, and striatum, and played an important role in physiological regulation processes such as neurogenesis and plasticity [21,22]. MiR-199a-5p prevented hypoxia induced injury of brain spinal cord and myocardium [3]. MiR-199a-5p promoted endogenous neurogenesis, improved neurological function after ischemic stroke, and reduce infarct volume [3,23,24].

Compared with healthy people, the expression of miR-199a-5p in the serum of patients with cerebral ischemia was obviously declined, which indicated that miR-199a-5p was essential in the process of cerebral ischemia [24,25]. MiR-199a-5p participated in regulating angiogenesis and vascular integrity by directly inhibiting the phosphatidylinositol-3 kinase regulatory subunit 2 (PIK3R2) [26], which was a negative regulator of the vascular endothelial growth factor (VEGF) pathway [26,27]. The expression of PIK3R2 had intimately correlation with Akt pathway [27,28].

Studies indicated that miR-199a-5p improved cognitive function and reduced hippocampal neuronal apoptosis in ischemic stroke rats by regulating the AKT signaling pathway [19]. These findings suggested that miR-199a-5p probably protected BBB integrity and reducing neuronal apoptosis after cerebral ischemia. Therefore, we hypothesize that miR-199a-5p could maintain BBB integrity and reduce neuronal apoptosis after ischemic stroke by regulating the Akt signaling pathway. The present study used the middle cerebral artery occlusion rats to explored the hypothesis.

Materials and methods

Ethics statement

All animal experiments were performed in strict accordance with the Guidelines for Animal Experiments at the second hospital of Hebei Medical University. The experimental protocols were approved by the Animal Experiments Committee of the second hospital of Hebei Medical University (Permit number: 2023-AE-200). All procedures were performed under isoflurane inhalation anesthesia, and every effort was made to minimize the suffering of the rats.

Animal and experimental groups

Adult male Sprague-Dawley(SD) rats (260−280g) used for the experiment was purchased from Weitong Lihua Laboratory Animal Technology (Beijing, China). The rats lived in cages which filled with sterile wood shavings as bedding material under 12/12 h light/dark cycle at 22°C± 3°C and 55% ± 10% relative humidity. Rats had free access to food and water. The experiment started after 7 days of adaptive feeding.

The SD rats were randomly divided into four groups: Sham group, MCAO group, miR-199a-5p group, miRNA-NC group.

Modeling and intervention of rats

Using middle cerebral artery occlusion (MCAO) was applied to induce ischemic stroke. The rats were anesthetized with 3.5% isoflurane. Following anesthesia, the right middle cerebral arteries of rats were occluded by way of inserting a 6–0 nylon monofilament (depth about18.5 ± 0.5mm). Laser Doppler blood flow measurement successfully ensured occlusion (MCA blood flow reduction exceeded 75%). Ligated the remaining end of the ECA, cut the redundant part of the nylon, and sutured the incision. After MCAO, temperature of the rats was maintained at 36.5–37°C. The rats in the Sham group were performed without any injury of arteries.

In the light of the Zea Longa Behavior Rating Scale, the neurological function was divided into five points: 0 indicated no neurological deficits; 1 point was lightly focal neurological deficits (the left front paw failed to fully extend); 2 points was classified as medium focal neurological deficits (leftward rotation); 3 points were serious focal deficits (tilted to the left); 4 points was unable to walk and had an inferior consciousness. Excluded MCAO rats with scores 0 and 4, and supplied in time.

Mixed separately miR-199a-5p mimic and negative control (Gemma Gene, Shanghai) with pure water(250μl) and dissolved. Transfection reagent Enterster TM in vivo (Ingen, Beijing) diluted with physiological saline, and 33μl Added 217μl physiological saline. The diluted in vivo transfection reagent mixed with microRNA yielded 10μl microRNA solution. The rats were fixed on a stereotactic frame. The solution was injected slowly into the right lateral ventricle of rats for 5 min (0.8mm posterior to bregma, 1.5mm lateral to midline, 3.5mm below the surface of the skull). In miR-199a-5p group and miR-NC group, rats were injected separately with miR-199a-5p mimic(5μl) and miR-NC(5μl). Injected equal volume of transfection agent into right lateral ventricle of rats in Sham group and MCAO group. After injection, left the needle with the syringe for another 2 minutes. Then removed the needle and sealed the hole with bone wax, sutured the skin, and immediately perform MCAO.

Neurobehavior assessment

Ludmila Belayev 12-point scoring (LB 12 Scoring) was applied to assess the sensorimotor integration function of rats 24 hours after MCAO. LB 12 Scoring included posture reflex test and limb placement test. The postural reflex test was a tail lift suspension test: 0 points for no neurological deficits, 1 point for normal limb flexion, and 2 points for positive pushing test. The limb placement test included: firstly, in the visual test, the researcher held the rat in his hand and suspended its forepaws. Placed the rat 10 centimeters over the table and approached the table slowly. The natural reaction of rats was to immediately grab the table with forelimbs, while MCAO rats moved slowly or could not accurately touch the table. 0 points: Normal response in rats; 1 point: Slow reaction, but not exceeding 2 seconds; 2 points: Slow reaction for more than 2 seconds. Horizontal stimulation, placed the rat above the desktop side, and followed the same procedure and scoring criteria as before. Tactile test included front and side stimuli. The rat’s eyes were entirely covered and its forepaws suspended in the air. The skin and hair of their forepaws gently touched the table. The response and scoring criteria of rats were identical with visual tests. Thirdly, the proprioception test, only involved anterior stimulation, manipulation and scoring criteria were identical with tactile test, which evaluated the sense of space, position and balance of rats. LB 12 Scoring ranged from 0 to 12 points. The more serious, the higher the score.

Tissue preparation

To avoid causing distress, the rats were euthanized with an overdose 3.5% isoflurane at 24 hours after neurobehavior test. Eye vein blood of rats were collected and serum was extracted, stored at -80°C. 0.1M phosphate buffer solution (PBS, pH 7.4) was perfused into through left ventricle. Subsequently, 4% paraformaldehyde was perfused for fixation. The right brain tissues were removed and separated infarct area and ischemic penumbra area, and then stored at -80°C.

Quantitative real-time PCR

Frozen serum and brain tissue were homogenized using ultrasound in 1ml triazole reagent. Incubated the homogenate at 25°C for 5 minutes to isolate the complex nucleoprotein. Then, added 0.2ml chloroform, shook the homogenate and incubated at 25°C for 5 minutes. Next, centrifuged the sample at 10000g at 4°C for 15 minutes. Mixed the aqueous phase including RNA with 0.5ml isopropanol, incubated at 25°C for 10 minutes, and centrifuged at 10000g at 4°C for 10 minutes. Resuspended RNA particles in 75% ethanol, centrifuged under 3500g at 4°C for 5 minutes, dried and dissolved in 20μl 0.1% DEPC water. 30 μl RNase-free ddH2O was added at 25°C for 2 minutes. Dissolved the precipitate thoroughly and obtained the total RNA of the sample, and then extracted it using Trizol reagent (Invitrogen Corp China).

Synthesized cDNA in the light of the M-MLV conserved transcriptase specification (Invitrogen Corp China). PCR reaction was performed using 1.5ml cDNA sample and SYBR Primex Ex Taq (TaKaRa, Kusatsu, Japan) in 20ml by the Exicycler 96 system (Bioneer, Daejeon, Korea). Calculated the relative expression by comparative 2-△△CT. All procedures were performed separately more than 3 times. The primers for RT-PCR were showed in Table 1.

10.1371/journal.pone.0306793.t001 Table 1 The primer information.

Gene	Forward Primer	Reverse Primer	
miR-199a-5p	5’-GCATCGTCGTACCGTGAGTAAT-3’	5’-GTGCAGGGTCCGAGGTATTC-3’	
Claudin-5	5’-TGGTGCTGTGTCTGGTAGGATGGA-3’	5’-GTCACGATGTTGTGGTCCAGGAAG-3’	
VEGF	5’-TCACCAAGGCCAGCACATAG-3’	5’-GGGCACCAACGTACACGC-3’	
β-actin	5’-GGCACCCAGCACAATGAA-3’	5’-AGAAGCATTTGCGGTGG-3’	

Western blotting

Expression levels of Claudin-5, VEGF, PIK3R2, P-AKT, and AKT in cerebral ischemic penumbra were measured by Western blotting. Homogenized the tissue in lysis buffer, which contained 10 μl inhibitor mixture in 500 ml RIPA buffer. After boiling with SDS for 5 minutes, the segregated proteins were electrophoretically on 8% polyacrylamide and then transferred to PVDF membrane. Incubated non-specific binding sites overnight in TBST (20mM Tris HCl, pH 7.61mM NaCl, 0.05%) at 4°C with anti Claudin-5 (1:500, Abbkine, USA), anti VEGF (1:500), anti PIK3R2 (1:1000, Boster, China), anti P-AKT (1:1000, Abcam, China), and anti AKT (1:500, Santa Cruz, USA). Washed 3 times with TBST, and incubated by goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, China) for 60 minutes. Immunoblotting was washed 3 times with TBST and emerged on X-ray film. Protein molecules size was decided through moving the protein ladder (Fermentas, Canada) in adjacent lanes. Image J software was used to scan and quantify film signals. Anti β-Actin (1:1000, Abcam, China) was an internal control and the relative protein levels were standardized to β-Actin.

Evans blue (EB) test of Blood brain barrier (BBB) permeability experiment

After neurological function assessment, 2% Evans blue dye (2mL/kg) was injected through the tail vein. After 2 hours, the rats were euthanized and treated with physiological saline from the left ventricle. The brain was excised, weighed, incubated at 37°C for 24 hours, centrifuged at 2000g for 10 minutes, collected the supernatant. Measured the absorbance value at a wavelength of 632 nm and calculated the content of Evans blue based on the standard curve.

Immunofluorescence staining

Frozen slices were roasted at 37°C for 30 minutes, then rinsed with PBS. sealed in a 37°C wet box with 2% BSA or 10% BSA for 30 minutes. Added appropriately diluted mouse anti NeuN (1:200, Abcam UK) to the sample slices, and diluted the first anti myeloperoxidase (MPO) (1:200, Beyotime, China) with PBS with a ratio of 1:200. Until the tissue was completely covered, and placed it overnight in a wet box at 4°C. Then, added Cy3 labeled IgG goat anti rabbit (1:200, Beyotime, China) secondary antibody, incubated in darkness at 37°C for 60 minutes, and rinsed three times with PBS. On the basis of the instructions of the Tunel cell apoptosis detection kit, prepared and dropped the reaction solution. Incubated in the dark at 37°C for 60 minutes, rinsed with PBS three times, stained with DAPI for 3 minutes, and sealed. Selected more than five slices from each group and observed them with fluorescence microscope. All images were captured by fluorescence microscope (Olympus, Tokyo, Japan). Analyzed the fluorescence intensity and number of neurons with Image Pro Plus software.

Statistical analysis

SPSS 22.0 computer software (SPSS, USA) was used to analyzed the data. All data were presented as Mean ± SD. Analyze multiple comparison procedures with one-way ANOVA. P-value <0.05 indicated statistical significance.

Results

miR-199a-5p mimic improved behavioral in MCAO rats

After 24 hours of cerebral ischemia and miRNA treatment, neurobehavior of MCAO rats were assessed by LB 12-Scoring. The results suggested that the LB 12-Scoring of MCAO rats were significantly reduced (P<0.05). Cerebral ischemic caused serious neurological lesion, which was obviously improved by miR-199a-5p mimic treatment. however, the rats in miR-NC group show opposite outcomes (Fig 1).

10.1371/journal.pone.0306793.g001 Fig 1 Ludmila Belayev 12-point scoring between groups.

MiR-199a-5p increased the mRNA expression of Claudin-5 and VEGF in the infarcted area and penumbra, reduced the lesion to the BBB of MCAO rats

RT-PCR was used for assessing the expression of miR-199a-5p, Claudin-5, and VEGF in serum, ischemic penumbra, and infarcted area. The results indicated that the expression level of miR-199a-5p and Claudin-5 were remarkably enhanced in serum, ischemic penumbra, and infarcted area (Fig 2A and 2B), at 24 hours after MCAO. MiR-199a-5p mimic treatment alleviated the lesion of cerebral ischemia to the BBB. Except for the Sham group, there was no noticeable difference in the expression of VEGF in the serum of MCAO rats among the other groups. The expression of VEGF in the Sham group was higher than those of cerebral ischemic rats (Fig 2C). The miR-199a-5p mimic obviously increased the expression of VEGF in the infarcted area and penumbra. Nevertheless, the expression of Claudin-5 and VEGF in the infarcted area and penumbra of miR-NC group were lower than MiR-199a-5p group (Fig 2A–2C).

10.1371/journal.pone.0306793.g002 Fig 2 Expression level of MiR-199a-5p, Claudin-5 mRNA, VEGF-A mRNA, in Serum, penumbra, and infarcted area between groups.

MiR-199a-5p protected the integrity of the BBB through regulating Akt signaling pathway

Western blotting was used to explore whether the miR-199a-5p maintained the integrity of BBB by regulating Akt signaling pathway. The expression levels of Claudin-5, VEGF, PIK3R2, p-Akt, and Akt were measured by Western blotting (Fig 3). The expression level of PIK3R2 was significantly increased following cerebral ischemic. Compared with miR-NC group, miR-199a-5p mimic treatment obviously reduced PIK3R2 expression (Fig 3C) and raised Claudin-5, VEGF expression (Fig 3A and 3B), and protected the integrity of BBB. Detecting the activation level of Akt in the penumbra area of cerebral ischemia, miR-199a-5p mimic markedly reversed the Akt inactivation caused by cerebral ischemia (Fig 3D). The data suggested that miR-199a-5p mimic protected the integrity of BBB following ischemic stroke.

10.1371/journal.pone.0306793.g003 Fig 3 Western blots and semiquantitative analysis of Claudin-5, VEGF-A, PIK3R2, P-Akt/Akt in cerebral ischemic penumbra between groups.

MiR-199a-5p decreased BBB permeability after cerebral ischemia in rats

The Evans blue penetration test was used to evaluate the permeability of BBB in rats (Fig 4). The exudation of Evans blue in the MCAO group was higher than that in the Sham group, that indicated the BBB permeability was enhanced after ischemia stroke. Compared with the MCAO group and miR-NC group, miR-199a-5p mimic clearly reduced cerebral vascular permeability after MCAO (Fig 4B). These results suggested that miR-199a-5p mimic exerted a protective effect on BBB integrity after cerebral ischemia.

10.1371/journal.pone.0306793.g004 Fig 4 Evans Blue extravasation between groups.

MiR-199a-5p inhibited leukocyte infiltration after MCAO

In order to investigate whether miR-199a-5p reduced leukocyte cells infiltration in the penumbra of MCAO rats, we detected the number of MPO+ cells using immunofluorescence staining (Fig 5). The outcomes indicated that the number of MPO+ cells in MCAO rats was dramatically boosted. Following miR-199a-5p mimic treatment, the number of MPO+ cells was remarkablely decreased, while the number of MPO+ cells in the miR-NC group was obviously increased. The results suggested that miR-199a-5p could inhibit leukocyte cells infiltration after MCAO (Fig 5A and 5B).

10.1371/journal.pone.0306793.g005 Fig 5 Quantification data of the MPO+ cells in cerebral ischemic penumbra between groups.

MiR-199a-5p lessened apoptosis in the ischemic penumbra of MCAO rats

Immunohistochemistry was used to detect the number of TUNEL+ cells in the ischemic penumbra of MCAO rats (Fig 6). Compared with the MCAO group and miR-NC group, the number of TUNEL+ cells in the miR-199a-5p group was significantly reduced, indicated that miR-199a-5p decreased apoptosis in the penumbra of cerebral ischemia (Fig 6A and 6B).

10.1371/journal.pone.0306793.g006 Fig 6 The rate of apoptosis cell in cerebral ischemic penumbra between groups.

MiR-199a-5p inhibited neuronal apoptosis in the ischemic penumbra of MCAO rats

NeuN+/TUNEL+ double stained cells were investigated by immunofluorescence (Fig 7). The results suggested that there were almost no apoptotic neurons in the Sham group. MCAO rats showed a large number of neuronal apoptosis in the ischemic penumbra. However, miR-199a-5p mimic significantly diminished neuronal apoptosis (Fig 7A and 7B).

10.1371/journal.pone.0306793.g007 Fig 7 The rate of apoptosis neurons in cerebral ischemic penumbra between groups.

Discussion

Blood brain barrier injury was an important pathological process in the pathogenesis of ischemic stroke, which probably disrupted the homeostasis of the brain environment, exacerbated inflammatory reactions, and caused neuronal apoptosis. The results of this study showed that after 24 hours of treatment with miR-199a-5p mimic, the limb sensory motor integration ability of MCAO rats was better than that of the MCAO group and miR-NC group. MiR-199a-5p reduced the expression of PIK3R2 protein in MCAO rats, enhanced Akt activation, obviously upregulated the gene and protein expression of Claudin-5 and VEGF in the infarcted area and ischemic penumbra, reduced BBB damage, and protected its integrity. Evans blue staining results showed that compared to the MCAO group and miR-NC group, miR-199a-5p significantly reduced the blood-brain barrier permeability in MCAO rats. Meanwhile, immunofluorescence staining results showed that miR-199a-5p inhibited leukocyte cells infiltration after cerebral ischemia, alleviated inflammatory, and diclined neuronal apoptosis in the ischemic penumbra area.

Neurological deficit score, improved Bederson score, and Longa 5 points method were commonly used for evaluating neurological deficits, but, they didn’t provide a detailed evaluation of the overall changes in neurological function. The Ludmila Belayev 12 point scoring method in this study comprehensively assessed the sensory motor integration ability of MCAO rats, and evaluate the neural function of MCAO rats through the combination of visual, tactile, proprioceptive, and motor functions, which were more convincing.

PI3K/AKT was a classic neuroprotective signaling pathway, and its mechanism of protecting neuronal cell survival mainly included: 1) inhibiting neuronal cell apoptosis by producing nerve growth factors and neurotrophic factors [29]; 2) Phosphorylation activated downstream substrates to inhibit cell apoptosis [30]; 3) Intervention of neuronal apoptosis through mitochondrial pathways [31]; 4) Protecting neurons through the erythropoietin and its receptor system. PIK3R2 was a member of the PI3K subunit family, which inhibited the activation of the PI3K/Akt pathway [32–34], regulated cell proliferation, migration, and maturation. Meanwhile, it was also a negative regulatory factor of VEGF [33–35]. MiR-199a-5p alleviated BBB damage, suppressed inflammatory reaction, and neuron damage caused by cerebral ischemia by inhibiting PIK3R2 and activating Akt.

BBB played a crucial role in regulating brain metabolism and maintaining central nervous system homeostasis. TJ proteins were the basis structural of BBB [13,15,36]. Claudin-5 was a key TJ protein that maintained the integrity and permeability of BBB, and its damage was the beginning of the disruption of BBB integrity in many pathological processes of brain injury diseases [15,37–39]. Vascular endothelial cells were a prominent structure that constituted the BBB, VEGF was a key regulatory factor for endothelial growth. Promoting the binding of VEGF to its receptors was great significance for protecting the integrity of BBB [40–42]. 24 hours after cerebral ischemia in rats, it was the peak period of brain edema and inflammatory reaction [24,43]. The expression level of TJ protein Claudin-5 was evidently diminished [6,35,44], and the permeability of BBB was increased. MicroRNA has been proven to alleviate inflammation and edema after cerebral ischemia, promote nerve regeneration and angiogenesis [33,45–47]. MiR-199a-5p intervention promoted the expression of Claudin-5 and VEGF, protect the integrity of BBB, and alleviated local inflammatory. Researches had found that miR-199a-5p was involved in regulating neuronal regeneration in the hippocampus and subventricular area after cerebral ischemia, and alleviated edema caused by cerebral ischemia. The results of this study suggested that miR-199a-5p cut down neuronal apoptosis in the ischemic penumbra, which could be closely related to the involvement of miR-199a-5p in reducing BBB permeability and alleviating local inflammatory. Other studies had shown that the level of Claudin-5 decreased in the early stages of vascular remodeling and increased in the later stages, and this dynamic change was closely related to the dynamic changes in BBB permeability [22,46–48]. In the present study, miR-199a-5p upregulated the expression of Claudin-5 and VEGF by regulating the PI3K/AKT signaling pathway, promoting endothelial neurogenesis in the ischemic penumbra and reducing the permeability of Evans blue.

Myeloperoxidase (MPO) was a functional and activation marker of neutrophils, a vital marker of inflammatory, and closely related to the inflammatory after cerebral ischemia [5,8]. MPO mediated high-density lipoprotein oxidation weaken endothelial cell proliferation and migration, and inhibited the activation of the Akt signaling pathway [26]. Bushueva et al. [27] found that the CpG site of the MPO gene in leukocyte cells of stroke patients undergone significant hypomethylation, exacerbating oxidative stress. MiR-199a-5p downregulated the expression of PIK3R2 and activated the Akt signaling pathway, alleviating inflammatory cell infiltration in the ischemic penumbra of MCAO rats.

Although the present study has achieved some meaningful results, there are still some limitations. Include the volume of cerebral infarction was not assessed in MCAO rats, and the brain water content was not measured before and after treatment in MCAO rats.

Conclusion

In summary, the findings of the present study indicated that miR-199a-5p inhibited inflammation following ischemic stroke by activating the PI3K/Akt signaling pathway, reduced neuronal apoptosis in the ischemic penumbra, and upregulated Claudin-5 and VEGF to preserve the integrity of the BBB. miR-199a-5p will be anticipated to serve as a novel therapeutic target for cerebral ischemia. However, this study still has limitations. While exploring the mechanism by which miR-199a-5p protected the BBB integrity after cerebral ischemia using miR-199a-5p mimics and negative controls, the role of endogenous miR-199a-5p cannot be fully ruled out.

In the future, the research team will investigate the mechanisms by which miR-199a-5p preserves BBB integrity following cerebral ischemia, either through silencing or using PI3K/Akt signaling pathway blockers, and the mechanisms by which miR-199a-5p promotes neurogenesis following ischemic stroke.

10.1371/journal.pone.0306793.r001
Decision Letter 0
Sessa Francesco Academic Editor
© 2024 Francesco Sessa
2024
Francesco Sessa
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
2 Apr 2024

PONE-D-24-00772MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathwayPLOS ONE

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"National Natural Science Foundation of China，（No.81273609）

Natural Science Foundation of Hebei Province，(No. H2023206115)"

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Additional Editor Comments:

The reviewers raised several important concerns. I believe that authors should further improve their manuscript. Please, revise the manuscript solving all criticisms and providing the rebuttal letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors studied the preventive and therapeutic effects of miR-199a-5p through PI3K/Akt pathway on ischemia stroke. On this basis, they further investigated the role of miR-199a-5p in BBB. Despite some highlights, this paper has a number of shortcomings, such as the logic of manuscript, the data etc, and page numbers and line numbers are missing.

1.The introduction lacks logic. Especially, it did not describe the relationship between miRNA and BBB. Please describe in detail.

2.Where are the tables? I can’t find them. Neurological function scores are better presented as bar graphs than table.

3.The improvement effect of miRNA on MCAO was only evaluated with neurological function score. It should add the data of infarct size and pathological status.

4.Although this study provides detailed evidence of the efficacy of CC, it does not provide in-depth mechanistic validation.

5.There is no logic in the results section. It should be showed from effect to mechanism.

6.Fig. 6A should add the merged figure.

7.The discussion lacks logic. Moreover, miR-199a-5p, as the key to this paper, has not been highlighted in the discussion.

8.Another obvious problem with this paper is lack of sufficient explanation of the results. More explanations on them seem necessary and helpful to readers.

9.The first paragraph of the discussion requires a brief summary of the research results.

10. It is noted that your manuscript needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure so that the goals and results of the study are clear to the reader.

Reviewer #2: This is a well-organized study exploring the association of MicroRNA-199a-5p attenuates BBB disruption following ischemic stroke.

Major:

The concept of the study is not novel and the topic itself is important, however, the lack of clear rationale and mechanistic assessments reduces the scientific value of the paper.

The English used in the manuscript is understandable but the text needs to be edited by a native speaker. Spelling, grammar, and syntax errors are present in the text. In the study implications, novelty aspects of the study and limitations are not indicated.

Minor:

please correct abbreviations - for example, in the introduction, CNS has already been used before, and TJs, not TJS

- access to food and not diet

- there are some misleading sentences in the text for example that miR-199a-5p was a main factor in the process of cerebral ischemia - introduction

**********

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Reviewer #1: No

Reviewer #2: No

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment Submitted filename: PONE-D-24-00772.docx

10.1371/journal.pone.0306793.r002
Author response to Decision Letter 0
Submission Version1
28 May 2024

Response to Reviewers

Dear Editors and Reviewers:

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathway”. (ID: PONE-D-24-00772). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to the research. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as following: Responds to the reviewer’s comments:

1.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

Response: A separate file labeled 'Revised Manuscript with Track Changes' has been uploaded.

2.Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Response : The funders "National Natural Science Foundation of China（No.81273609)" had a role in study design and preparation of the manuscript. The funders " Natural Science Foundation of Hebei Province (No. H2023206115)" had a role in data collection and analysis, decision to publish.

4. For example, authors should submit the following data: The values behind the means, standard deviations and other measures reported; The values used to build graphs; The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

Response : The data has been upload as a separate file labeled " date of graphs and figures" has been uploaded.

Reviewer #1: In this manuscript, the authors studied the preventive and therapeutic effects of miR-199a-5p through PI3K/Akt pathway on ischemia stroke. On this basis, they further investigated the role of miR-199a-5p in BBB. Despite some highlights, this paper has a number of shortcomings, such as the logic of manuscript, the data etc, and page numbers and line numbers are missing.

Response: Considering the Reviewer’s suggestion, we have added page numbers and line numbers.

1.The introduction lacks logic. Especially, it did not describe the relationship between miRNA and BBB. Please describe in detail.

Response: thank you for your suggestion. We have revised the introduction and described the relationship between miRNA and BBB.

2.Where are the tables? I can’t find them. Neurological function scores are better presented as bar graphs than table.

Response: The tables were uploaded as separate file. Neurological function scores have been presented as bar graphs.

3.The improvement effect of miRNA on MCAO was only evaluated with neurological function score. It should add the data of infarct size and pathological status.

Response: thank you for your suggestion. In the present study, we primarily investigated the protective effect of miR-199a-5p on the BBB in MCAO rats through regulating PI3K/Akt signaling pathway. Thus, the measurement of cerebral infarction volume was not considered in the study design.

4.Although this study provides detailed evidence of the efficacy of CC, it does not provide in-depth mechanistic validation.

Response: In our study, the efficacy of CC was not validated, and it was not mentioned in the manuscript.

5.There is no logic in the results section. It should be showed from effect to mechanism.

Response: In the results section, the results of the experiment were presented, and the relevant mechanisms are presented in the discussion section.

6.Fig. 6A should add the merged figure.

Response: In Fig. 6A, the left side showed the staining of neuronal nuclei, and the right side showed the result of NEUN+/TUNEL+ staining, which was the merge figure.

7.The discussion lacks logic. Moreover, miR-199a-5p, as the key to this paper, has not been highlighted in the discussion.

Response: Thank you for the reviewer's comments. As per the reviewer's recommendations, the discussion has been revised. Special emphasis was placed on miR-199a-5p.

8.Another obvious problem with this paper is lack of sufficient explanation of the results. More explanations on them seem necessary and helpful to readers.

Response: Thank you for the reviewer's suggestion. In the discussion section, a detailed explanation of the results was provided.

9.The first paragraph of the discussion requires a brief summary of the research results.

Response: Thank you for the reviewer's comments. A brief summary of the research results was presented in the first paragraph of the discussion section.

10. It is noted that your manuscript needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure so that the goals and results of the study are clear to the reader.

Response: We appreciate the reviewer's suggestions. The manuscript has been revised by professionals to correct the grammar, tense, and sentence structure in the text, making it easier to read.

Reviewer #2: This is a well-organized study exploring the association of MicroRNA-199a-5p attenuates BBB disruption following ischemic stroke.

Major:

The concept of the study is not novel and the topic itself is important, however, the lack of clear rationale and mechanistic assessments reduces the scientific value of the paper.

The English used in the manuscript is understandable but the text needs to be edited by a native speaker. Spelling, grammar, and syntax errors are present in the text. In the study implications, novelty aspects of the study and limitations are not indicated.

Response: We appreciate the reviewer's suggestions. The findings of the present study indicated that miR-199a-5p inhibit inflammation following ischemic stroke by activating the PI3K/Akt signaling pathway, reduced neuronal apoptosis in the ischemic penumbra, and upregulated Claudin-5 and VEGF to preserve the integrity of the BBB. miR-199a-5p will be anticipated to serve as a novel therapeutic target for cerebral ischemia.

The manuscript has been revised by professionals to correct the grammar, tense, and sentence structure in the text, making it easier to read.

Reviewer #2: This is a well-organized study exploring the association of MicroRNA-199a-5p attenuates BBB disruption following ischemic stroke.

Minor:

please correct abbreviations - for example, in the introduction, CNS has already been used before, and TJs, not TJS

- access to food and not diet

-there are some misleading sentences in the text for example that miR-199a-5p was a main factor in the process of cerebral ischemia - introduction

Response: We appreciate the reviewer's attention to detail. We have made revisions one by one according to the suggestions.

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0306793.r003
Decision Letter 1
Sessa Francesco Academic Editor
© 2024 Francesco Sessa
2024
Francesco Sessa
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
18 Jun 2024

PONE-D-24-00772R1MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathwayPLOS ONE

Dear Dr. NI,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: Despite Reviewer#1 rejecting the manuscript, in my opinion, the authors improved the manuscript substantially. I suggest revisiting it and inserting a separate section with the study limitations (the authors should insert the limitations described by reviewer#1). Please, insert it before conclusion: in this way, it could be published.

==============================

Please submit your revised manuscript by Aug 02 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Francesco Sessa, Ph.D., MS

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Despite Reviewer#1 rejecting the manuscript, in my opinion, the authors improved the manuscript substantially. I suggest revisiting it and inserting a separate section with the study limitations (the authors should insert the limitations described by reviewer#1: "Although the authors have made numerous modifications for the manuscript, the current data do not support the conclusions. The data of infarct size and pathological status are important index for evaluating ischemic stroke. They are not provided.").

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Although the authors have made numerous modifications for the manuscript, the current data do not support the conclusions. The data of infarct size and pathological status are important index for evaluating ischemic stroke. They are not provided.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10.1371/journal.pone.0306793.r004
Author response to Decision Letter 1
Submission Version2
21 Jun 2024

The study limitations have been inserted before conclusion.Xiaoqing Wang was listed in the authorship.

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0306793.r005
Decision Letter 2
Sessa Francesco Academic Editor
© 2024 Francesco Sessa
2024
Francesco Sessa
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
25 Jun 2024

MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathway

PONE-D-24-00772R2

Dear Dr. NI,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Francesco Sessa, Ph.D., MS

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors modified the manuscript following the reviewers' suggestions.

Reviewers' comments:

10.1371/journal.pone.0306793.r006
Acceptance letter
Sessa Francesco Academic Editor
© 2024 Francesco Sessa
2024
Francesco Sessa
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
27 Jun 2024

PONE-D-24-00772R2

PLOS ONE

Dear Dr. NI,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Lecturer Francesco Sessa

Academic Editor

PLOS ONE
==== Refs
References

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2 W Andres A Rothstein H Elser , et al . Trends in the Prevalence of Stroke Among Community-Dwelling Individuals in the US, 1999–2018. Jama neurol. 2023; 80 :646–648. doi: 10.1001/jamaneurol.2023.0742 37094376
3 Zhong W ,Li Y-C ,Huang Q-Y , et al . lncRNA ANRIL ameliorates oxygen and glucose deprivation (OGD) induced injury in neuron cells via miR-199a-5p/CAV-1 axis. Neurochemical research. 2020; 45 :772–782. doi: 10.1007/s11064-019-02951-w 31907708
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5 Tu W-J ,Chao B ,Wang L . Prevalence of stroke in China: overestimated? The Lancet Public Health. 2022; 7 :e404. doi: 10.1016/S2468-2667(22)00066-4 35487227
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7 Tian DS ,Liu CC ,Wang CL , et al . Prevalence and risk factors of stroke in China: a national serial cross-sectional study from 2003 to 2018. Stroke Vasc Neurol. 2023; 8 :238–248. doi: 10.1136/svn-2022-001598 36418056
8 Zhang G ,Pan Y ,Zhang R , et al . Prevalence and prognostic significance of malnutrition risk in patients with acute ischemic stroke: results from the third China national stroke registry. Stroke. 2022; 53 :111–119. doi: 10.1161/STROKEAHA.121.034366 34645284
9 Delfino C ,Nuñez M ,Asenjo-Lobos C , et al . Stroke in Latin America: Systematic review of incidence, prevalence, and case-fatality in 1997–2021. International Journal of Stroke. 2023: 18 :645–656. doi: 10.1177/17474930221143323 36428236
10 Xiong Y ,Fu Y ,Li Z , et al . Laquinimod Inhibits Microglial Activation, Astrogliosis, BBB Damage, and Infarction and Improves Neurological Damage after Ischemic Stroke. ACS Chem Neurosci. 2023; 14 :1992–2007. doi: 10.1021/acschemneuro.2c00740 37161270
11 Lee MJ ,Zhu J ,An JH , et al . A transcriptomic analysis of cerebral microvessels reveals the involvement of Notch1 signaling in endothelial mitochondrial-dysfunction-dependent BBB disruption. Fluids and barriers of the CNS. 2022; 19 :1–15.34983574
12 Hu D ,Mo X ,Luo J , et al . 17-DMAG ameliorates neuroinflammation and BBB disruption via SOX5 mediated PI3K/Akt pathway after intracerebral hemorrhage in rats. International Immunopharmacology. 2023; 123 :110698. doi: 10.1016/j.intimp.2023.110698 37517381
13 Hou W ,Yao J ,Liu J , et al . USP14 inhibition promotes recovery by protecting BBB integrity and attenuating neuroinflammation in MCAO mice. CNS Neurosci Ther. 2023; 29 :3612–3623. doi: 10.1111/cns.14292 37269080
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