Neuroprotection of Extracellular Vesicles From Human Adipose Stem Cells Intranasally Administered 24 Hours After Stroke In Rats


 Ischemic stroke is a prominent cause of death and disability, demanding for innovative and reachable therapeutic strategies. Thus, approaches presenting optimal period for therapeutic intervention and new routes of treatments administration as promising tools for stroke treatment. In this study, we evaluated the potential neuroprotective properties of nasally administration of human adipose tissue stem cells (hAT-MSC)-derived extracellular vesicles (EVs), obtained from healthy individuals submitted to liposuction. A single intranasal EVs (200 µg/Kg) administration performed 24 h after a focal permanent ischemic stroke in rats. Was observed a higher tropism of EVs by peri-infarct zone, surrounding the infarct core. Interestingly, in the same brain region, was observed a significant decrease in the volume of the infarct, improvement the blood-brain barrier and re-stabilization of vascularization. In addition, EVs recover the impairment on long-term motor and behavioral performance induced by ischemic stroke. Surprisingly, one single intranasal EVs administration reestablished: i) front paws symmetry; ii), short- and long-term memory; iii) anxiety-like behavior. In keeping with this, our work highlights hAT-MSC-derived EVs as a promising therapeutic strategy in stroke.

The ischemic stroke pathophysiology is characterized by blood ow obstruction of a restricted brain region, forming an infarct nucleus, surrounded by an area known as penumbra region. Of note, this region has the potential to be reperfused [11] and in animals models refers as the peri-infarct region [12].
Reperfusion of the penumbra/peri-infarct zone contributes to a reduction in the nal infarct size and attenuation/reversal of neurological and behavioral de cits [13,14]. Therefore, therapeutic strategies with focus on penumbra region salvage are intensively searched [15]. Currently, the gold standard treatment for ischemic stroke is the use of thrombolytic agents, which focuses on optimizing the reperfusion time in the penumbra region [16]. However, this strategy has to be strictly applied within 4.5 h after the rst symptoms [17]. In addition, reperfusion injury, such as hemorrhage, is the most dangerous collateral effect after thrombolysis [18,19]. Thus, the thrombolytic treatment is contraindicated for a certain class of patients at risk for bleeding or for the formation of large blood clots [20]. Additionally, some patients would be considered for endovascular therapy (Mechanical Thrombectomy), which could increase the maximum time to receive treatment up to 24h after rst symptoms; however, this procedure requires quali ed professionals and infrastructure to conduct imaging exams (computed tomography scan and angiogram computed tomography) [18,21]. Therefore, it is essential to nd new and accessible therapeutic strategies for ischemic stroke. In vivo studies using rat models of brain ischemia demonstrated that treatment with mesenchymal stem cells (MSCs) increased the therapeutic window up to 24 h after insult [22][23][24]. Though MSCs therapy seems very promising, it may cause some impacting damage such as immune system rejection and increased risk of developing tumor tissue [25]. In this way, improvements in MSC therapy have been made to optimize its e cacy. It is currently thought that MSCs protective effects are due to the release of extracellular vesicles (EVs). The EVs are small double membrane vesicles (30-200nm), released by many cell types, which in physiological conditions mediate cell-to-cell communication [26]. Compared to MSCs, EVs have lower immunogenicity, decreasing the risk of obstructive vascular effect and of secondary microvascular thrombosis and presenting higher ability to cross the blood-brain barrier (BBB) [27,28].
However, EVs systemically administered may undergo metabolization before reaching the brain tissue [33], being detected in other organs (such as lungs, liver and spleen) [34]. Thus, some studies are focusing on a straightforward and noninvasive strategy to administer EVs targeting the brain, as a potential strategy to treat brain disorders [35][36][37].
In this study, we investigated the neuroprotective effects of EVs released from human adipose tissue mesenchymal stem cell (hAT-MSCs), through a single intranasal administration 24 h after ischemic injury in rats. In speci c, we evaluated short-and long-term effects of a permanent focal stroke model and the EVs effects on forepaws symmetry, behavioral performance, anxiety-like behavior, brain infarct volume, BBB permeability and brain formation of new blood vessels.
Materials And Methods 1. hAT-MSCs: sources, culture and characterization.
The cells were obtained from commercial and human sources 1a. Commercial hAT-MSCs.
Flow cytometry: hAT-MSCs were centrifuged (400×g for 5 min at room temperature), the cell pellet was resuspended in DMEM + 10% FBS and the cells were counted in a Neubauer chamber. Shortly after, cells were incubated with antibodies at concentration of 1:50 for 4 h at 37°C. Then, cell suspensions were centrifuged at 400×g for 5 min at room temperature, and cell pellets resuspended in 200 µl of PBS. Ten thousand events were analyzed using ow cytometry (BD FACSCalibur™) [40]. Cells, only in passage 4 (P4), were characterized as hAT-MSCs by CDs presence: CD34 (FITC Mouse Anti-human CD34 BD Pharmingen), CD45 (Human CD45 FITC Conjugate, Invitrogen), CD90 (PE Mouse Anti-Human CD90 BD Pharmingen) and CD105 (Huan CD105 R-PE conjugate, Invitrogen).
Confocal microscopy: an aliquot of 1x10 4 hAT-MSCs was placed on a slide and analyzed by immuno uorescence. Cells were maintained in culture conditions for 72 h to adhere to the coverslip. Then, cells were incubated for 4 h at 37°C with the same antibodies used for cytometry: CD34, CD45, CD90 and CD105, at a ratio of 1:500. The negative control was prepared by incubating only the secondary antibodies: Alexa Fluor 555 (Invitrogen) and Alexa Fluor 488 (Invitrogen). Cells were gently washed in the coverslip with PBS (4 times) to remove excessive antibodies, followed by xation with PFA 4% for 2 h. Cells were gently washed again with PBS, and the coverslips xed with Fluoromount (Sigma) onto a histological slide for further analysis. Images were acquired using an 8-bit gray scale confocal laser scanning microscope (Olympus FV1000). Approximately 10−15 sections with 0.7 µm thick confocal were captured parallel to the coverslip (XY sections) using a ×20 objective (Olympus, U plan-superapochromat, UPLSAPO 60X). Z-stack reconstruction and analysis were conducted using ImageJ software (http://rsb.info.nih.gov/ij/).

Extracellular Vesicles (EVs)
2a. EVs isolation and puri cation As cultured hAT-MSCs (P4-P8) reached 80% con uence, DMEM + 10% FBS medium was replaced by DMEM FBS-free (to avoid isolated vesicles contamination by FBS proteins). After 72 h of culture, the medium was collected for vesicles isolation and cells remained in culture. To recover from stress caused by FBS removal, the remained cells were supplemented with DMEM + 10% FBS for 72 h [29].
For EVs isolation, the medium was collected and centrifuged (3 times) at 4°C: (1st : 400×g for 15 min, 2nd : 2000×g for 15 min and 3rd : 10,000×g for 30 min). The supernatants were ltered through a 0.22 µm membrane. The isolation was nished by centrifugation (100,000×g at 4°C for 2 h). The supernatant was discarded, PBS was used to wash the pellet containing EVs, and the cell suspension was centrifuged at 100,000×g at 4°C for 2 h [41]. Finally, the pellet was resuspended in 100 µl of PBS and stored at − 20°C [38]. EVs protein content was quanti ed by bicinchoninic acid (BCA) assay (Thermo Scienti c Pierce™) [38]. The vesicles isolated from C0, C1 and C2 cells are named EV0, EV1 and EV2, respectively.

2b. EVs Characterization
EVs were characterized by ow cytometry and identi cation of membrane proteins CD63 and CD81 presence [42]. Firstly, EVs were incubated with magnetic beads (Thermo Fisher -Scienti c -Invitrogen™) coated with primary antibody CD63 (Exosome-Human CD63, Thermo Fisher -Scienti c -Invitrogen™) and CD81 (Exosome-Human CD81, Thermo Fisher -Scienti c -Invitrogen™) for 18 h at 4°C under gentle stirring. For each preparation, 10 µl of 1 mg/ml EVs suspension was applied. To remove excess of beads, EVs were washed with PBS: 2mL of PBS was added for 5 minutes, then the tub was placed in a magnet for 1 minute and the supernatant was discarded. Then, CD63 (CD63 Anti-human Mouse, FITC, Clone: MEM-259, Invitrogen ™) and CD81 (PE Anti-Human Mouse CD81 Clone JS-81, BD Pharmingen ™) antibodies (without granules) were added to the solution containing the EVs + magnetic beads. After 1 h of incubation, the EVs were gently washed by placing the tube on a magnet for 1 minute, discarding the supernatant. We added 2mL of PBS (to remove excess antibody) for 5 minutes and again placed the tube on a magnet for 1 minute and discarded the supernatant. Finally, the EVs were resuspended in 200 µl of PBS for analysis. Ten thousand events were analyzed by ow cytometry.
To measure the particle size and the polydispersity index (PDI) we used photon correlation spectroscopy. The EVs suspension derived from hAT-MSCs (50 µl) at 1 mg/ml was diluted in 1 ml of PBS. All analyzes were performed in triplicate using a Malvern Nano-ZS90® (Malvern Instruments, England) at 25 ° C.

2c. EVs Purity Measurement
Transmission electron microscopy (TEM) analysis, using direct examination technique, was used to evaluate EV purity and diameter sizes [37]. EVs suspension (10 µL), 1 mg/ml of protein, was aliquoted onto a grid covered with carbon lm (formvar/carbon) and dried at room temperature. Uranyl was used as a contrast. The sample was analyzed by TEM 120Kv (JEM 1200 Exll-JEOL).

2d. EVs Labeling
EVs were labeled with red uorescent membrane dye PKH26 (MINI26, Sigma). In brief, the EVs-containing PBS solution was centrifuged at 100,000×g for 2 h, at 4°C and the pellet suspended with the diluent of the uorescent kit. Filtered PKH26 (4mM) and EVs (200 µg/ml) were mixed at a ratio 1:1 for 5 min, followed by the addition of 5% BSA. To remove excess dye, the EVs were washed (3 times): we added 5 ml of PBS and centrifuged at 100,000×g for 2 h, at 4°C, discarded the supernatant. In the last centrifugation, stained EVs pellet was suspended in 0.

3b. Focal Permanent Ischemia and Sham Procedures
Anesthetized animals (ketamine hydrochloride: 90 mg/kg, 450 µl/kg i.p. and xylazine hydrochloride:10 mg/kg, 300 µl/kg i.p.) were placed into a stereotaxic apparatus. After skin incision, the skull was exposed, and the craniotomy was performed by exposing the left frontoparietal cortex (+ 2mm to − 6mm A.P. and − 2mm to − 4mm M.L. from the bregma). A focal permanent ischemic lesion was induced by thermocoagulation of motor and sensorimotor pial vessels [43][44][45][46][47]. Blood vessels were thermo-coagulated by placing a hot probe near the dura mater for 2 min, until red-brown color indicated complete thermocoagulation. Soon after, the skin was sutured, and animals placed on a heating pad at 37°C, until full recovery from anesthesia. Animals from sham group were only submitted to the above-mentioned craniotomy. Animals were randomly allocated to 3 treatment groups: Sham; Ischemic (ISC); Ischemic treated with EVs (ISC + EV).
Slices images for counting EVs were acquired using an 8-bit gray scale confocal laser scanning microscope (Olympus FV1000). Approximately 10−15 sections with 0.7 µm thick confocal were captured parallel to the coverslip (XY sections) using a ×60 objective (Olympus, U plan-super-apochromat, UPLSAPO 60X). Z-stack reconstruction and analysis to count the vesicles in the brain tissue were conducted using ImageJ software, brie y: background noise was removed using the "subtract background" tool. Images were converted to binary masks using default threshold option and vesicles were counted with the "analyze particles" tool (size = 0.05-0.90 µm). These settings were programmed into a macro and used for all analyzed images (http://rsb.info.nih.gov/ij/) (n = 3 naive and n = 5 ischemic for each group, 3 sections in each rat per group).

4b. Short term infarct volume
For the short-term evaluation of infarct volume, Naive, Sham, ISC, ISC + EV0 and ISC + EV2 groups were sedated 48 hours after treatment (O 2 ow rate of 0.8 -1.0 mL/min with Iso urane levels of 2.5-3.0%) and culled. Coronal sections of the whole brains were sliced at 2 mm, immersed in 2% 2,3,5-Triphenyltetrazolium chloride (TTC). After 30 minutes of incubation at 37 ºC, the slices were dipped in 4% buffered paraformaldehyde (pH 7.4) for 24 hours. The size of the infarct area was evaluated as the area devoid red staining. Infarct volume was measured using the imageJ software [44] (3 rats/group, 6 sections/rat).

4c. Brain Angiogenesis
After 42 days of treatment, animals from groups Naive, ISC and ISC + EV2 were anesthetized and received intracardial injection of 50 mg/mL (500 µl) uorescein isothiocyanate-dextran amine (Merck) to label brain blood vessels. Rat brains were removed, immediately xed in PFA 4%, and cut at 30 µm coronal slices in a vibratome. Images were acquired in uorescence microscope (Nikon). The images were taken from the ipsilateral and contralateral sides in the Secondary Motor Cortex (M2) and somatosensory regions (SS) using the coordinates: +2.20 mm, 0.2 mm and − 1.88 mm A.P. to Bregma (PAXINUS online Rat Brain Atlas: http://labs.gaidi.ca/rat-brain-atlas/) (n = 3 per group). Blood vessels parameters as the total length (sum of length of segments, isolated elements and branches in the analyzed area) and the number of branches (in the analyzed area) were quanti ed with the Angiogenesis Analyser Plugin (Gilles Carpentier Research) ImageJ software (https://imagej.nih.gov/ij/).

BBB permeability
5a. Evans Blue in brain parenchyma.
Naive (n = 3), Sham (n = 3), ISC (n = 3) and ISC + EV2 (n = 3) animals were anesthetized 48 hours after treatment (ketamine hydrochloride -90 mg/kg, 450 µl/kg i.p. and xylazine hydrochloride 10 mg/kg, 300 µl/kg i.p.) and received 3 ml/kg of Evans Blue (EB) solution 2% in saline, through the gingival artery (Supplementary information 1). After 1 hour, the animals were submitted to cardiac perfusion using a peristaltic pump (10 mL/min, with PBS, 100mL). Animals were culled, their brains were removed, weighed and whole brain was sliced at 2 mm for image acquisition for each slice. After, all slices together from each brain were macerated and homogenized in 2.5 ml of PBS and vortexed for 2 minutes. For the precipitation of proteins, 2.5 ml of 50% trichloroacetic acid was added to the homogenate, incubated for 12 hours at 50 ºC, centrifuged at 14,000 x g for 10 minutes. The concentration of the blue color was measured by a spectrophotometer (620 nm). EB dye was expressed in µg/g brain tissue against a standard curve [48,49].

5b. CSF albumin levels
Albumin assay was performed using High Performance Liquid Chromatography coupled to Fluorescence detector (HPLC-FLD). The CSF method was validated according with the FDA guidelines [50,51]. HPLC-FLD consisting of LC Shimadzu system (Kyoto, Japan) equipped with LC-20AT pump, DGU-14A degasser, thermostat for CTO-10A column and Fluorescence detector, RF 20A was used. Acquisition and processing of the data was obtained using the LC Solution software. The FLD was set at 278 nm (excitation) and 335 nm (emission). Agilent reversed-phase ZORBAX SB-C18 column (5 µm particle size, 250 × 4.6 mm i.d.) was used. The method was performed using gradient condition, consisting of solvent A (H 2 O + 0.1% formic acid) and solvent B (acetonitrile (ACN) as follows: A → 65% B → 35% (0-5 min), A → 70% B → 30% (5-10 min), A → 65% B → 35% (10-17 min). The ow rate was set at 0.7 mL/min. Samples preparation was performed by adding to a 10 µL liquor in 40 µL of ACN and mixed in a vortex. The solution was transferred to conical vials and 10 µL was injected. Albumin stock solutions 1 mg/mL in water were stored at − 20 ± 2°C. For each day of analysis, standard solutions of albumin were prepared at 0,1, 0,5, 1, 10, 50 and 100 µg/mL.

Behavioral evaluation 6a. Cylinder Task (CT)
The cylinder task, which allows the evaluation of motor sequelae caused by ischemic insult [52], were used to determine animal motor symmetry of front paws. Exploration of the apparatus by the rats was evaluated when they raised their bodies and contact their paw(s) on the cylinder wall (20 movements are counted). The apparatus consisted of a transparent glass cylinder 20 cm in diameter and 30 cm in height. All animals were submitted to this task 2 h before surgery, to verify the basal forelimb symmetry. The CT was repeated on the 3rd, 7th, 14th, 21st, 28th, 35th and 42nd days after EVs treatment. The performance was recorded using ANY-Maze software (Stoelting CO., Wood Dale, IL), and the ipsilateral (to the lesion), contralateral or both front paws preference were counted in a blind analysis. The asymmetry of each animal was calculated by the following formula: asymmetry = (% of ipsilateral paw use = Ipsilateral paw use / sum Ipsilateral + Contralateral + use of both paws) − (% of contralateral paw use = contralateral paw use / sum of Ipsilateral + Contralateral + use of both paws). The asymmetry percentage was converted into symmetry percentage [43]. Groups: Naive (n = 6), ISC (n = 22), ISC + EV0 (n = 17); ISC + EV1 (n = 16) and ISC + EV2 (n = 17). At the end of each task, the apparatus was cleaned using 70% ethanol solution.

6b. Open Field Task (OFT)
The open eld task evaluates habituation to novelty (assessing short-and long-term memoryexploratory activity) and locomotor activity in an arena [53]. The apparatus consisted of a black cage measuring 50 cm in length × 50 cm in width × 50 cm in height. The sessions lasted 10 min (individually). Animals performed the task on the 7th, 21st and 42nd days after EVs treatment. Short-term memory (habituation to novelty) was evaluated considering the decrease of locomotion during the rst 5 min of the 1st session (7th day). Long-term memory was evaluated considering the decrease in locomotion during the rst minute through the successive sessions (from the 1st to the 3rd session). Groups: Naive (n = 8), Naïve + EV0 (n = 7), ISC (n = 22), ISC + EV0 (n = 17); ISC + EV1 (n = 16) and ISC + EV2 (n = 17). At the end of each session, the apparatus was cleaned with 70% ethanol solution. The task was recorded and analyzed using ANY-maze 6.1 software.

6c. Novel Object Recognition Task (NORT)
The behavioral sessions lasting 10 min were performed on the 7th, 21st and 42nd days after EVs treatment. 90 min after the OFT session, Object Recognition (OR) short-and long-term memories were evaluated [54]. Animals were individually placed on the periphery of the arena for exploration. Two identic familiar objects (FOs) were placed in the arena and animals were allowed to explore them for 10 min. Sni ng and touching the objects were considered as exploratory behavior. 90 min after the training session, each animal was placed back into the arena to evaluate short-term memory. One of the 2 FOs used in the training session was replaced by a new distinct object (NO). The long-term memory was evaluated 24 h after the short-term memory task session, when the animals were placed back in the arena with same FO used in the training session and in rst test session (short-term memory), while the same NO was displaced to a different position. In all sessions, the time spent exploring the objects was recorded by using ANY-maze 6.1 software. Results were expressed as a percentage of time exploring each object. Animals that recognized the novel object (short-term memory) or its new position (long-term memory), explored it more than 50% of the total exploring time of both objects. Groups: Naive (n = 8), Naive + EV0 (n = 7), ISC (n = 22), ISC + EV0 (n = 17); ISC + EV1 (n = 5) and ISC + EV2 (n = 16). At the end of each session, the apparatus was cleaned using 70% ethanol solution.
6d. Elevated Plus-maze Task (EPMT) The EPMT task is widely used to study anxiety-like behavior [55]. The apparatus had 2 open arms (50 cm long × 10 cm wide) and 2 closed arms (50 cm long × 10 cm wide × 40 cm high), separated by a central platform (5 cm long × 5 cm wide). The apparatus was placed 70 cm high from the oor. The animals were kept in a red-light area for 1 hour before starting this task. ANY-maze software was used to record behavioral performance for 5 min. The percentage of time spent in open and closed arms was assessed. Anxiety-like behavior was considered as the increase of time spent on closed arms. Each animal conducted this once, on the 7th day after treatment with EVs. At the end of each session, the equipment was cleaned with 70% alcohol.

Statistical Analysis
The size of the brain lesion, BBB integrity, number of vesicles found in brain tissue and angiogenesis analysis were evaluated by unpaired T-tests. Two-way RM ANOVA was applied for CT, followed by Sidak's multiple comparisons test. Short-term memory was evaluated by unpaired t-test. Long-term memory was evaluated by two-way ANOVA followed by Sidak's multiple comparisons test. Unpaired T-tests were used for the NORT with a theoretical average of 50%. Data are reported as the mean ± SD. All analyses were performed using Graph Pad Prism 6.0.

hAT-MSCs and EVs
1a. hAT-MSCs Characterization Figure 2. There is no single marker to characterize hADSCs, which is done by immunophenotyping based on the presence (> 70%) of CD90 and CD105, associated with the absence (< 5%) of CD34 and CD45 [29]. Displacement of the uorescence peaking to the right side registers a positive value for protein markers presence. More than 70% of C1, C2 and C3 cells were positive for CD90 and CD105, while only 0.3% of the analyzed cells were positive for CD45 and CD34, which is a characteristic of these cells. The cells were also characterized by uorescence microscopy with the same markers.
1b. Extracellular Vesicles (EVs) Characterization, Figure 3. EVs were detected inside the C0 cells by confocal microscopy through the presence of CD63 and CD81 marking (Fig. 3a, 3b and 3c). They were detected in the plasma membrane and in the cytoplasm near to the nucleus. The released EVs (EV0, EV1 and EV2) were analyzed by measuring CD63 and CD81 marking through ow cytometry; more than 90% of the EVs presented CD63 and CD81 [42] (Fig. 2d). The released EVs (EV0, EV1 and EV2), were also analyzed by Zetasizer instrument, which indicated the average diameter of 140 nm, with a polydispersity index (PDI) average of 0.3 (Fig. 3e). The purity of released EVs suspension was con rmed by the TEM-direct technique. The suspension only consisted of released vesicles with cylindrical morphology and electron dense membranes (indicated by the black arrow in Fig. 3f).

Stroke damage and EVs neuroprotection 2a. EVs administration on motor neuroprotection
Dose curve of EV1 administration and motor neuroprotection Figure 4. The cylinder task (CT) measures front paw symmetry and was applied to identify the lowest dose to be used for EV stroke treatment. All animals underwent the CT task 24 h before stroke (day − 1). Of note, only animals presenting ~ 100% front paw symmetry were included in the study. Further, we performed intranasal administration of EV0 or vehicle 24h after stroke. Indeed, 72 h after stroke, all ISC groups, treated or not, presented a mean symmetry of 30%. Interestingly, animals treated with EVs showed gradual improvement in symmetry of the front paws compared to untreated animals in a time and dose dependent manner: at the 42nd day after intranasal administration, animals in the vehicle and treated EVs (100µg/kg, 200µg/kg or 300µg/kg) groups presented symmetry recovery of 58% ±6, 67% ±4%, 87% ±8 and 82% ±3, respectively. Animals receiving 200µg/kg or 300 µg/kg did not differ from naive group from the 21st day after treatment with VES (95% ±3), suggesting a total recovery of symmetry. In keeping with this, the dose of 200 µg/kg at 24 h after stroke was selected for the further experiments.

2c. BBB permeability
As illustrated in Fig. 7, stroke affected the BBB permeability, an effect partially attenuated by EVs treatment. These ndings were demonstrated by Evans blue dye penetration into brain parenchyma ( Fig. 7a and 7b) and by an increase of CSF albumin levels (Fig. 7c) (ISC x ISC + EV3, p < 0.05).
2d. Extracellular Vesicles Detection (EVs) in Rat Brain 2e. Open Field Task (OFT) Figure 9. This task evaluates the habituation to novelty. Animals were submitted to 3 sequential OFT sessions: in the 7th, 21st and 42nd days after EVs treatment. All groups presented short-term memory (evaluated only in the 1st exhibition). Naive groups also presented long-term memory, which was impaired by stroke; EV0, EV1 and EV2 treatment abolished this effect.
2f. Novel Object Recognition Task (NORT) Figure 10. The NORT was used to evaluate short-and long-term memory of object recognition (OR) on the 7th, 21st and 42nd days after EV0, EV1 or EV2 treatment. Stroke impaired both short-and long-term memory, an effect abolished by EVs treatment.
2g. Elevated Plus-maze Task (EPMT) Figure 11. The elevated plus-maze task is widely used to evaluate anxiety-like behavior. The task was performed on the 7th day after treatment with EVs. ISC animals spent more time on the closed arms compared to other groups, indicating that stroke induced an anxiogenic-like effect. Interestingly, the anxiety like behavior due to stroke was completely abolished by EV0 and EV1 treatment.
2h. Brain angiogenesis Figure 12. Stroke decreased the number of branches and the total length of blood vessels in the cortical M2-I region, (Fig. 12b and 12c). The reduction on the number of branches was abolished by EVs treatment. Figures 12d-12i show representative images of the M2 regions. In SS regions, there was no difference among all groups (Supplementary information 2).

Discussion
The two available strategies for stroke treatment are the use of thrombolytic agents and the mechanical removal of the thrombus. Nevertheless, in both cases accessibility may be limited. In fact, thrombolytic agents have to be strictly applied within 4.5 h after the rst symptoms [15], while specialized equipment and highly trained people are required to remove the thrombus [19]. In keeping with this, the search for innovative and more accessible treatment strategies for stroke are extremely relevant.
In this work, we demonstrated for the rst time that the use of intranasal hAT-MSC-derived EVs offers a broader early intervention opportunity (24 hours after the stroke), pointing to a potentially valuable stroke treatment strategy. Although there are studies showing neuroprotective effects of MSC-derived EVs on stroke [30,32,34], most of them apply systemic administration, a protocol in which EVs are detected in other organs than the brain, as lungs, liver and spleen [32,33], where they may be metabolized before reaching the cerebral parenchyma [56]. In these studies, the EVs were identi ed in brain regions but no study compared the number of EVs among regions [37,56,57] thus the EVs tropism for speci c brain regions was not previously reported.
Indeed, our ndings demonstrated a non-homogenous distribution in the ischemic group: ipsi-and contralateral M2 peri-infarct regions contained more EVs, compared to the SS regions (and also compared to the M2 and SS regions of naïve animals), indicating a higher EVs tropism to peri-infarct region. These results are in accordance with various previous works, which have focused on stroke treatment strategies targeting the peri-infarct region, aiming to stimulate angiogenesis [58,59] and to modulate the BBB permeability [33,59].
Clinical data from stroke patients have shown that behavioral and motor impairment are dependent on the regions of the brain where the infarct core and the penumbra zone are developed [3]. Accordingly, our and other research groups have already shown that in the ischemic stroke rat model here used, the core and peri-infarct regions are located in the prefrontal cortex and hippocampus [43][44][45][46][47], brain structures involved in neuromotor and memory modulation [60,61]. Here, our stroke model caused a localized BBB impairment (acutely measured by Evans blue) and a decrease in the vascularization (chronically measured), both speci cally in peri-infarct regions. Interestingly, a higher EVs tropism was observed to the same peri-infarct regions where a BBB recovery and vascularization improvement were demonstrated. This association points to a potential role of hAT-MSC derived EVs in brain located tissue repair, thus promoting motor and behavioral recovery.
In fact, stimulation of angiogenesis has been show to improve neurologic and motor function in animal stroke models [62,63], an effect currently acknowledged as outcome of EVs transfer of protein, mRNA and miRNA to endothelial cells [64], regulating proteins expression [65]. The BBB impairment in ischemic stroke is also documented [66], but its involvement in EVs therapeutic strategies was not previously reported.
We believe that our work shed light into a new and straightforward therapeutic strategy for focal permanent stroke treatment, by utilizing hAT-MSCs from healthy individuals as a source for EVs. In addition, the intranasal hAT-MSCs-derived EVs administration 24 h after brain injury induced a long-term neuroprotective effect, offering a remarkable broader therapeutic time window, compared to current standard systemic routes. Together, these ndings point to a potential therapeutic strategy for patients with focal permanent ischemic stroke. The authors declared no potential con icts of interest with respect to the research, authorship, and/or publication of this article.

Availability of data and material
The authors assume the availability of data and materials

Author contributions
All authors contributed in all stages of this work, read and approved the nal manuscript.
Compliance with ethical standards The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All procedures with animals were performed according to the Guide for the Care and Use of Laboratory Animals and to the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal studies. The Ethics Committee for the Use of Animals at the Universidade Federal do Rio Grande do Sul (process number: 31888) approved this project. The animals were followed up after the surgery, if signs of pain, discomfort, in ammation, and any other symptoms that indicate the animal's suffering were observed, after the surgery, it was euthanized, thus avoiding its suffering (Humanitarian endpoint).

Consent to participate
All participants who donated adipose tissue for cell isolation signed a free and informed consent form as recommended and approved by the Research and Graduate Group (Grupo de Pesquisa e Pós-Graduação: GPPG 2018-0374) and Research Ethics Committee (Comite de Ética em Pesquisa CAEE -: 94521618.4.0000.5327) of the Experimental Research Center at Hospital de Clínicas de Porto Alegre.
This document described the entire objective of the research and that these data could be published in a scienti c journal maintaining the con dentiality of the participants' personal data (anonymous donation) Consent for Publication: Not applicable.