Extracellular vesicles derived from astrocytes pretreated with melatonin promoted neuro-angiogenesis in mice with ischemic medial prefrontal cortex

Abstract

Background

Ischemic stroke (IS) is one of the leading causes of death and long-term disability worldwide. In addition to physical deficits, stroke survivors also exhibit cognitive impairment. Reparative properties of mouse astrocyte extracellular vesicles (AS-EVs) have been proven in ischemic conditions. Here, the neuro-angiogenesis properties of melatonin (MT)-astrocyte EVs (MT-AS-EVs) were studied in IS mice.

Methods

A total volume of 5 µl (~ 1 × 10¹¹ particles) of MT-AS-EVs and AS-EVs was administered into the penumbra of the ischemic medial prefrontal cortex induced by photothrombosis. After 21 days, brain samples were taken, and protein levels of neuroangiogenesis (NeuN and vWF) and synaptogenesis (Homer, Synaptophysin, and Synapsin) were assessed using immunofluorescence (IF) staining and western blotting. The proinflammatory response was monitored by measuring TNF-α and TGF-β in brain samples. Behavioral tests were also conducted to evaluate cognitive performance.

Results

From TTC staining confirmed the efficiency of the present protocol for inducing IS in the target region. The injection of AS-EVs, especially MT-AS-EVs, reduced the infarcted area compared to IS mice. IF staining revealed induction of vWF + and NeuN + cells at the ischemic site in mice treated with AS-EVs and MT-AS-EVs, with a significant effect (p < 0.05). The protein levels of Homer, Synaptophysin, and Synapsin increased in the presence of MT-AS-EVs compared to the other experimental group. Additionally, MT-AS-EVs had the potential to attenuate the pro-inflammatory response by reducing TNF-α and TGF-β levels compared with IS mice (p < 0.05). It was noted that the improvement in behavioral parameters in the IS mice after injection of EVs, especially MT-AS-EVs (p < 0.05).

Conclusions

AS-EVs are valid biological agents for inducing brain healing under IS conditions. Preconditioning with melatonin can enhance the reparative properties of AS-EVs, particularly in neuro-angiogenesis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-026-07709-x.

Introduction

Ischemic stroke (IS) is the most prevalent reason for human disability, with profound challenges to the healthcare systems [1, 2]. The recanalization approach is a significant breakthrough in IS individuals; however, it can be applied to a small fraction of patients [3]. Following IS, different underlying mechanisms can exacerbate the intensity of brain injury and lead to secondary damage. The accumulation of free radicals, lack of normal vascularization, and neurogenesis, neuronal excitotoxicity, and mitochondrial dysfunction occur post-IS [4]. With the brain parenchyma, the ischemic core, which irreversibly changes, such as neuronal and glial cell necrosis, and apoptosis, is surrounded by the penumbra, where the cells can survive for a limited period [5, 6].

Recent data have indicated that different brain regions, such as the prefrontal cortex (PFC), can be at risk of IS [7]. PFC encompasses lateral, medial, and orbitofrontal regions that can regulate several cognitive skills and functions such as decision-making, planning, social interaction, etc [8]. Recent advances show that the medial PFC (mPFC) plays a crucial role in many cognitive functions [9, 10]. Notably, mPFC is juxtaposed to subcortical areas (thalamus, amygdala, and hippocampus) with a particular top-down executive control over various cognitive areas [11, 12]. Depression and anxiety are common in IS survivors, resulting from direct ischemic injury to the mPFC [13]. It has been shown that the occurrence of depression in IS patients is closely associated with cognitive decline and delays the recovery process, and increases socioeconomic burden [13, 14].

In numerous preclinical IS models, movement disorders have been studied because these deficits are more easily studied in animal models, so improved preclinical ischemic models are needed to develop and optimize treatments for post-stroke depression (PSD) [15]. Unfortunately, middle cerebral artery occlusion (MCAO) is the most commonly used approach to induce focal IS in rodents, primarily affecting sensory-motor circuits and precluding assessment of cognitive functions [16, 17]. The photothrombotic method can, in part, overcome these limitations by inducing ischemic changes. This modality enables us to precisely target specific areas of the brain parenchyma involved in cognitive functions, without compromising motor function. mPFC damage can predispose to significant functional impairments and loss of cognitive and emotional processing [18].

For many years, recombinant tissue plasminogen activator (r-tPA) has been the only treatment available to eliminate blood clots and restore blood flow to the brain. However, this method has a narrow therapeutic window and is effective only up to about 4 h after the IS [19], making it necessary to develop new drugs and treatments. Several cell therapies and cell-based products that have entered the preclinical or early clinical phases have proven to be safe and effective for treating this disease in recent years [20]. In various studies, extracellular vesicles (EVs) have been found to have therapeutic properties for the acceleration of the healing process under IS conditions [21–23]. Either in physiological or pathological conditions, EVs can cross several natural barriers, such as the blood-brain barrier (BBB), and enter the brain due to their tiny size and reciprocal interaction with vascular cells [24]. By transferring bioactive molecules between nearby and distant cells, EVs act as a key component of the central nervous system (CNS) microenvironment. These vesicles can be used both as communication vehicles within and between different types of cells [25, 26]. EVs, as a key element of the microenvironment of the CNS, maintain intercellular communication via the transfer of bioactive molecules between different neural cells [27], and recent evidence suggests that EVs can orchestrate the neuro-angiogenesis [28]. In the nervous system, astrocytes are the primary glial cell type and are involved in brain tissue homeostasis, such as glial-mediated angiogenesis, nerve regeneration, neural progenitor cell migration, and glial plasticity via the release of several cytokines [29, 30]. Therefore, the control of astrocyte function, especially secretome via EVs, is thought to be a strategic approach in the control of neuro-angiogenesis during several pathological conditions [31, 32].

Over the past three decades, many studies have investigated the potential link between melatonin and CNS injury [33]. Melatonin is a hormone secreted by the pineal gland in the mammalian brain. Its earliest identified role is in regulating circadian rhythms and the sleep-wake cycle [34]. The new findings have emphasized the role of melatonin in regulating many essential physiological and pathological functions. Melatonin is a highly efficient scavenger that removes hydroxyl radicals and hydrogen peroxide and has a neuroprotective role in neurological injuries such as ischemic stroke and traumatic brain injury [35, 36]. Melatonin, with anti-inflammatory, antioxidant, and neuroprotective effects in neuropathophysiological models, plays an indispensable role in neural cell function [37]. Therefore, this study aims to address the issue of whether EVs from astrocytes pre-treated with melatonin can influence neural regeneration and improvement of cognitive functions in IS mice (Fig. 1).

Fig. 1.

Schematic illustration of the experimental process. Astrocytes were isolated from the mouse brain, characterized, and then treated with melatonin. In the next step, the EVs were extracted, and a photothrombotic focal ischemia model was induced in mice. Following stroke induction, mice received EVs, and behavioral and histological analyses were then performed to evaluate neuro-regeneration and behavior. Created by BioRender’s web-based software

Materials and methods

Animal ethics

8- to 12-week-old male BALB/c mice, weighing 22–25 g, were used in the current experiment. Animals were kept in the animal house of the Neuroscience Research Center with a 2 h/12 h light/dark cycle at 25 °C and relative humidity ranging from 40 to 70% under standard conditions. Animals were accommodated in the Animal Houses for about 7 days before starting the experimental procedure. Mice were allowed to access chewing pellets and tap water ad libitum. All phases of this study were approved by the local ethics committee of Tabriz University of Medical Sciences (IR.TBZMED.AEC.1402.020) and the Vice President of Scientific Technology and Knowledge Council for the Development of Regenerative Medicine and Stem Cells Technologies. In this study, 40 mice are randomly divided into 4 groups. Control; Sham; IS + Astrocyte EVs (AS-EVs); IS + Melatonin-treated Astrocyte EVs (MT-AS-EVs).

Astrocyte isolation and expansion

The heads and necks of postnatal mice (P2–P4) were held and sprayed with 70% ethanol. The olfactory bulbs, cerebellum, and meninges were removed while dissecting the cerebral cortex. Astrocytes were isolated as follows: cortical tissues were chopped into tiny pieces with sharp blades, centrifuged for 5 min at 1500 g, and cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO₂. When the cells reached 80–90% confluency, the astrocytes were purified by initially shaking at 37 °C, 100 rounds/minute for 30 min to remove oligodendrocytes, and then shaking at 37 °C, 300 r/min for 3 h to remove microglia. After dissociating using 0.5% Trypsin-EDTA, cells were centrifuged for 5 min at 1500 g and seeded into T25 culture flasks [38].

Immunofluorescence (IF) staining

Astrocytes at passage three were fixed with 4% paraformaldehyde (PFA) for 20 min, washed with phosphate-buffered saline (PBS), and incubated with 0.3% Triton-X100 solution (Cat no: T8787; Sigma-Aldrich) for 30 min to permeabilize the cell membrane. After PBS washes, 10% goat serum (Cat. no. G9023; Sigma-Aldrich) was used to block nonspecific antibody binding. Thereafter, samples were incubated with anti-glial fibrillary acidic protein (GFAP; dilution: 1:100, Cat no: orb10706; Biorbyt Ltd) overnight at 4 °C. The next day, samples were washed several times with PBS and incubated with fluorochrome-conjugated secondary antibody (Dilution: 1:150; Cat no: orb-688925; Biorbyt Ltd) at RT for 1 h. The samples were again washed with PBS and incubated with DAPI (Cat no: D9542; Sigma-Aldrich) for nuclear staining. Samples were imaged using a fluorescent Olympus microscope.

EVs extraction

Cells at passages 3–6 were cultured in FBS-free culture medium for 72 h. The supernatants were subjected to different centrifugation speeds, including 400 g, 2000 g, and 10,000 g for 5, 10, and 30 min, respectively, to eliminate cells, debris, and subcellular organelles according to a previously described protocol [39]. Upon passing through a 0.22 μm microfilter, EV pellets were achieved by ultracentrifugation at 100,000 g for 60 min. The EV pellets were resuspended in PBS and stored at -80 °C until use [40].

Dynamic light scattering (DLS)

The hydrodynamic diameter and zeta potential of AS-EVs were measured using a Zetasizer instrument (Malvern Nano-Zetasizer) [41].

Scanning electron microscopy (SEM)

SEM was used to measure the morphology and non-hydrodynamic diameter of isolated EVs. In brief, EVs were fixed using 4% PFA and diluted in distilled water. Samples were placed on aluminum foils and allowed to air-dry, followed by dehydration using ascending EtOH concentration (40, 60, 80, 96, 98, and 100%). Samples were lyophilized, gold-sputtered, and imaged using an SEM device (TESCAN MIRA3) [42].

Western blotting

The presence of CD63, TSG101, and CD81 was confirmed by western blotting to verify the endosomal origin of isolated EVs. Total protein contents were extracted using RIPA protein lysis buffer, and about 10 µg of protein samples were separated on 10% SDS-PAGE electrophoresis. After transferring onto PDVF membranes and blocking with 5% non-fat milk (Sigma-Aldrich), a panel of primary antibodies, including CD63 Cat no: sc-5275; Santa Cruz Biotechnology Inc.), CD81 (Cat no: sc-166029; Santa Cruz Biotechnology Inc.), and TSG101 (Cat no: sc-7964; Santa Cruz Biotechnology Inc.), was used. After several PBS washes, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at RT. Using X-ray films and ECL solution, the immunoreactive bands were detected [43].

MTT assay

To determine the maximum melatonin dose with reduced astrocyte toxicity, an MTT assay was performed. The survival rates of astrocytes were determined in the presence of different concentrations of melatonin (0,10, 20, 40, 80, and 100 µM) after 24, 48, and 72 h. In short, astrocytes were resuspended in DMEM/F12 medium containing 10% FBS and 1% Pen-Strep, then transferred into 96-well plates. After the incubation time was complete, the supernatant was replaced with 20 µl of MTT (5 mg/ml). The plates were maintained at 37 °C for 3 h. The procedure continued with the addition of 100 µl of DMSO, and finally, the ODs were read at 560 nm using a microplate reader [44].

EV labeling

To track administrated AS-EVs inside the brain, isolated EVs were incubated with 20 µM CellTracker™ CM-DiI Dye (Cat no: C-7000; Invitrogen) for 20 min at 37 °C. After the staining process, EV pellets were collected by centrifugation at 100,000 × g at 4 °C for 60 min. Dil-labeled EVs were resuspended in 5 µl PBS solution and injected into the lateral ventricle of mice. In this study, mice received single doses of AS-EVs containing ~ 10¹¹ particles per 5 µl of protein.

Photothrombosis stroke induction

Mice were anesthetized by an injection of Ketamine (80 mg/kg)/ (10 mg/kg) Xylazine. After shaving the mouse skull hair, a longitudinal incision was induced down the center of the skull using a surgical blade, exposing the coronal and sagittal sutures. Based on Paxions and Watson’s stereotaxic atlas and the anterior-posterior (AP) and mediolateral (ML) coordinates for the mPFC (AP: +1 mm, ML: 0.5 mm), sterile Rose Bengal (20 g/body weight) was injected via IP and allowed for circulation for 5 min before starting the photothrombotic stroke induction. Next, the light-green laser was irradiated onto the skull surface for 10 min, and the wound was sutured. After surgery, mice were maintained at a controlled temperature. Three days after IS induction, to evaluate the effectiveness of this protocol for the induction of IS, we randomly selected mice, sacrificed them by an overdose of ketamine and xylazine, removed their brains, coronal sections were taken at identical intervals, stained with 2,3,5-triphenyl-tetrazolium chloride (TTC) solution (Sigma-Aldrich), and underwent behavior tests. After validating the animal model, EVs were injected into the penumbra of the ischemic lesion in the mPFC. In the last step, BrdU (Cat. no. 19–160; EMD Millipore Corporation) was injected daily for 3 days, until day 5.

Histological analyses

Immunofluorescence (IF) staining

On the 21st day after EV injection, mice were euthanized by an overdose of ketamine and xylazine, brains were removed, placed in a 4% PFA solution, and coronal sections were prepared at equal intervals. The expression of neuronal differentiation marker (NeuN) and angiogenesis factor [von Willebrand factor (vWF)] was monitored using IF staining. 5 μm-thick slides were permeabilized using 0.3% Triton X-100 solution, washed with PBS, and blocked using 10% goat serum for 1 h at RT. Then, samples were incubated with vWF primary antibodies (Cat. no. orb-158717; Bio-Rad) overnight at 4 °C. After that, slides were washed with PBS three times (each for 5 min), and incubated with FITC-conjugated secondary antibody (orb-158717; Biorbyt Ltd) for 1 h at RT. To stain nuclei, samples were stained with DAPI (Sigma-Aldrich) for 1 min. The percentage of vWF⁺/DAPI^_ cells was calculated in 10 high-power fields (HPF). For double BrdU/NeuN staining, samples were incubated overnight at 4 °C with anti-mouse BrdU (Dilution: 1:50; orb-625179; Biorbyt Ltd) and anti-mouse NeuN (Dilution: 1:100, Cat no: GTX638198; GeneTex) at 4 °C overnight. After washing with PBS, Alexa Fluor 488-conjugated secondary antibody was used for 2 h at RT. DAPI was used to stain nuclei, and the percentages of BrdU⁺/NeuN⁺ cells were calculated in at least 10 HPF.

Crystal Violet staining

Twenty-one days of EV injection, three mice per group, underwent stereological analyses. The animals were euthanized by ketamine-xylazine overdose and perfused using normal saline, followed by the infusion of 4% PFA solution dissolved in 0.01 M phosphate buffer. The tissue was dehydrated through a graded ethanol series, cleared, and embedded in paraffin. Utilizing systematic uniform random sampling, twelve serial coronal sections were prepared and stained with 0.1% Cresyl violet.

Hematoxylin and Eosin (H&E) staining

Paraffin-embedded blocks were cut into 5 μm sections and stained with H&E. General features of tissue, such as inflammation, cell death, and histological changes, were followed in the stained slides and compared in different experimental groups [45].

ELISA assay

The concentration of TGF-β and TNF-α was determined using a mouse TNF-α ELISA kit (DY510-05, R&D) and a mouse TGF-β ELISA kit (DY1679-05, R&D), following the manufacturer’s instructions, respectively [46].

Monitoring synaptogenesis using Western blotting

Brain samples were lysed in RIPA lysis buffer, and total protein contents were measured using the BCA assay. Samples were electrophoresed (10% SDS-PAGE), transferred onto PVDF membranes, blocked using slim milk and incubated with anti-Homer (Dilution: 1:2000; Cat no: sc-17842- Santa Cruz Biotechnology Inc.), anti-synapsin (Dilution: 1:1000, Cat no: E-AB-33003; Elabscience Biotechnology), and anti-synaptophysin (Dilution: 1:500, Cat no: sc-17750; Santa Cruz Biotechnology Inc.) antibodies [47]. The density of immunoreactive bands was calculated using ImageJ software (Ver. 1.4).

Behavioral assessments

Before mice euthanasia, several cognitive tests, including measuring anxiety, depression, and sensory-motor disorders, were performed on day 21 after injection. Mice were placed in normal lighting conditions (white light). All animals were of the same age at the start of the experiment. The assays follow as;

Tail suspension test (TST)

The TST is a widely used behavioral assessment for screening antidepressant drugs and evaluating the effects of treatments on depression-like behaviors. In this assay, mice were suspended by their tails with adhesive tape, preventing escape and contact with nearby surfaces. During the six-minute trial, we measured the latency and number of escape-oriented movements, such as struggling or climbing attempts, as indicators of behavioral despair or resilience [48].

Light/dark transition test

Anxiety-like behavior can be measured using the light/dark transition test, which is based on mice’s natural aversion to brightly illuminated areas and spontaneous exploration of novel environments. The apparatus has a dark and brightly lit chamber, and mice can freely move between the two. The number of times mice entered the bright chamber and their duration were indices of their bright-space anxiety. After 30 min, mice are transferred to the behavior testing room and allowed to freely move between the two chambers with the door open for 10 min [49].

Force swim test (FST)

The FST is a behavioral test that assesses the efficacy of potential antidepressant treatments. Depressive-like behavior was evaluated by placing mice in a water-filled cylinder (height: 60 cm, water depth: 40 cm, temperature: 24 °C ± 1) for a 6-minute test session. The sessions were recorded on video, and three specific behaviors (swimming and immobility) were evaluated using a time-sampling method. Swimming involved horizontal movements within the cylinder, including diving, and immobility was characterized by a floating, upright posture with minimal movements to stay above the water [50].

Open field test (OFT)

The OFT is used to measure locomotion, hyperactivity, and some aspects of anxiety-related behavior in rodent models. Locomotion was measured in an open field test setup consisting of four adjacent, individual arenas (100 × 100 × 80 cm). Animals were placed in the center of the square and allowed to move freely for 5 min. The sessions were video-recorded, and the total distance traveled and average speed were calculated by tracking the center point of the mice’s body contour using ESTraS software [51].

Statistical analysis

Data (mean ± SD) were compared between groups using ANOVA with Tukey post hoc analysis (GraphPad Prism Ver. 10). Statistical significance was adjusted to p < 0.05.

Result

AS-EVs isolation and characterization

Using the mechanical method, mouse AS-EVs were isolated by dissecting the cerebral cortex, chopping the cortical tissue, and culturing the isolated cells under standard conditions. Phase-contrast microscopy revealed that primary astrocytes displayed polygonal, fusiform, and flat morphologies (Fig. 2A). The data showed that the typical astrocyte marker GFAP was expressed in nearly 71.23% of cultured astrocytes at passage 3, demonstrating the efficiency of the current protocol for isolating mouse brain target cells (Fig. 2B). Western blotting confirmed the existence of tetraspanins (CD63 and CD81), along with ESCRT complex factor (TSG101), in the isolated AS-EVs (Fig. 2C). SEM data showed the heterogeneous spherical EV particles (Fig. 2D). DLS data revealed a hydrodynamic diameter of 329 ± 26.43 nm and a 32.3 mV zeta potential in AS-EVs (Fig. 2E-F). These data demonstrate that the isolated particles are identical to AS-EVs and the Exo subset.

Fig. 2.

Characterization of primary astrocytes and their derived extracellular vesicles (AS-EVs). Bright-field images indicate the polygonal to the fusiform shapes of primary astrocytes at passage 3 (A). Immunofluorescence staining confirms astrocytic identity through positive expression of the glial fibrillary acidic protein (GFAP) marker, indicating high culture purity (B). Western blot analysis demonstrates the presence of EV protein markers (C), SEM images reveal the spherical morphology of isolated AS-EVs (D), DLS analysis shows the size distribution profile of AS-EVs, indicating a mean diameter (E), and zeta potential of AS-EVs that reflects their colloidal stability in suspension (F)

Melatonin dose optimization

To determine the optimal dose of melatonin for astrocyte preconditioning, an MTT assay was used to assess astrocyte survival at 24, 48, and 72 h after exposure to various melatonin concentrations (Fig. 3A-C). Based on the data, the maximum survival rates were achieved at three time points (24, 48, and 72 h) in the group that received 40 µM melatonin compared to the control cells (p < 0.05). Increasing the melatonin dose to more than 40 µM did not yield a statistically significant difference compared to the control group. Specifically, treatment of mouse astrocytes with 80 µM or 100 µM melatonin resulted in suboptimal survival rates. In addition to these data, a 40 µM melatonin dose was selected for astrocyte preconditioning and further analyses.

Fig. 3.

Figure 3. Monitoring the survival rate of primary mouse astrocytes after being treated with different doses of melatonin for 24 (A), 48 (B), and 72 hours (C). Data represent mean ± SD, n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test (*p < 0.05; **p < 0.01). Tracking of Dil-labeled AS-EVs in the subventricular zone (SVZ) region of the mouse brain (D). Data indicate the internalization of red-colored AS-EVs inside the cytosol of green-colored Nestin+ neural stem cell (NSCs). Nuclei were stained using DAPI. Scale bar = 100 µm. One-way ANOVA with Tukey post-hoc analysis. *p<0.05; **p<0.01; . Mouse brain after IS, Intact brain. Triphenyl tetrazolium chloride (TTC) stained brain section 72 hours after IS induction, representative (E). Data demonstrate that the infarcted region appears pale due to the lack of vascularization (the restricted area outlined in black), contrasting with the deep red color of viable/less affected tissue in the brain's cortex. tail suspension test (TST) (F) and forced swim test (FST) (G) showed a significant increase in immobility time correlates with depressive behavior in IS mice. Student-t test (n=10). ****p<0.0001

EV uptake by mouse neural stem cells (NSCs)

To track injected AS-EVs in the brain parenchyma, EVs were stained with red CM-DiI. The IF data confirmed the colocalization of Dil-labeled EVs into Nestin⁺ NSCs in the SVZ region (Fig. 3D). These data indicate the efficient delivery of labeled AS-EVs into the neurogenic niche in the mouse brain.

Induction and validation of photothrombotic IS

To validate ischemic changes, TTC staining was performed 72 h after photothrombotic modulation of IS induction. Based on our data, the ischemic brain zone appeared pale and sharply outlined in the cortical region, while the neighboring viable region was red due to a regular vascular bed (Fig. 3E; the restricted area outlined in black). Behavioral assessments were conducted to evaluate neurobehavioral impairment associated with the photothrombotic IS model. In the TST, the model animals displayed a significant increase in time spent immobile, and these mice tended to spend more time immobile in the FST, confirming impairment in neurobehavioral output (Fig. 4F-G; p < 0.0001). Taken together, these data confirmed the efficacy of our protocol for inducing a cortical IS model.

Fig. 4.

Histological evaluation of the ischemic mPFC region following the administration of AS-EVs and MT-AS-EVs using H&E staining (A) and Cresyl Violet staining (B). Brain sections are presented at three magnifications (4X, 10X, and 40X from left to right, respectively). Treatment with AS-EVs partially preserved tissue architecture, while MT-AS-EVs further improved histological outcomes, reducing necrotic areas and promoting organized cell morphology, suggesting neuroprotective effects. The MT-AS-EVs group had significantly less infarct volume and retention of cortical architecture

Histological changes of mPFC after AS-EVS administration

H & E staining revealed potential neuroprotective effects of AS-EVs, especially MT-AS-EVs, after administration at the ischemic site (Fig. 4A). In the sham group, severe ischemic lesions are evident in the granular and molecular layers of the cerebellar cortex, with gliosis (Fig. 4A). Data confirmed neuronal death, and most cells within the ischemic area exhibit pyknotic nuclei, characterized by small, dark-stained, hyperchromatic nuclei with disparate chromatin patterns. It was shown that AS-EV administration reduced cortical structural loss and pathological changes in the infarct-adjacent zone more than did the sham group. Histological observations demonstrated greater therapeutic benefits of MT-AS-EV transplantation into the ischemic site, presumably due to their greater potential to deliver antioxidative, anti-apoptotic, and pro-survival factors, which may help decrease ischemia-associated cytopathology and afford neurons greater protection. In the MT-AS-EVs, neuronal morphology is quietly improved, accompanied by a reduced acute inflammatory response.

Neuronal integrity was improved by MT-AS-EVs

Cresyl Violet staining showed widespread neuronal dropout, disrupted laminar structure, and large, cavitated infarct cores, with the largest infarct volume in the sham group (Fig. 4B). The complete loss of Nissl substance, combined with pale staining, indicated extensive neuronal degeneration. Nissl staining was paralleled by significant loss of NeuN⁺ mature neurons and very minimal BrdU incorporation, suggesting there was both severe neurodegeneration and very little neurogenesis at the cellular level (Fig. 4B). In the AS-EVs group, the infarct volume was moderately reduced with partial preservation of the cortical structure and increased density of neurons in the peri-infarct area. Cresyl Violet showed a more defined cortical architecture than the sham group. In the MT-AS-EV-treated group, greater tissue preservation and cellular regeneration, with limited cavitation, intact cortical layering, and numerous intact neurons, were achieved. Overall, these data indicated that MT-AS-EVs enhance neuroprotection and neuroregenerative properties by reducing structural infarct volume and promoting endogenous repair systems at the cellular level.

MT-AS-EVs enhanced vascularization in the ischemic mPFC region

The angiogenic potential of AS-EVs and MT-AS-EVs was assessed based on the local density of vWF⁺ vessels (Fig. 5). In the control group, a continuous capillary network with dense, vertically oriented vWF expression, indicating an active, intact, and robust endothelial barrier, is prominent. In the sham group, fewer vWF + vessels were observed due to ischemic changes induced compared to the control group (p < 0.05). The injection of AS-EVs and MT-AS-EVs into the ischemic mPFC led to a significant increase in vWF⁺ vessels compared to the sham group, indicating the induction of local angiogenesis and an increase in cortical vasculature after ischemic changes (Fig. 5). Notably, no statistically significant differences in vWF + vessels were observed between the AS-EVs and MT-AS-EVs groups. Despite the increase in vWF + vessels following administration of AS-EVs and MT-AS-EVs, these values were lower than those in the control group (p < 0.05). These data confirmed the angiogenic potential of both MT-EVs and AS-EVs in restoring angiogenesis in the ischemic mPFC region.

Fig. 5.

IF staining of vWF⁺ vessels to assess vascular integrity post-stroke in the mPFC (A-B). Control samples show robust vWF expression, while in the sham group, vWF⁺ vessels were significantly reduced (A-B). Treatment with AS-EVs and MT-AS-EVs can restore local angiogenesis and the number of vWF + vessels near control levels (n = 3). One-way ANOVA with Tukey post-hoc analysis. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001

MT-AS-EVs induced neurogenesis in the ischemic mPFC region

The neurogenic response of AS-EVs and MT-AS-EVs was assessed by BrdU and NeuN staining following IS (Fig. 6). Data indicated that NeuN⁺ cells were significantly reduced compared to the control group (p < 0.05), suggesting a considerable loss of mature neurons (Fig. 6). Besides, BrdU⁺ nuclei in the sham group were found in only a few regions, indicating poor endogenous proliferative activity following the ischemic injury (Fig. 6). The lack of BrdU/NeuN co-localizations suggests that even the few proliferating cells in the sham group are unlikely to differentiate into mature neurons under these circumstances. This phenomenon highlights the limited spontaneous regenerative abilities of the adult brain after IS. In the AS-EVs group, the number of NeuN⁺ cells was higher than in the sham group (Fig. 6). Along with these data, the number of BrdU⁺ was also elevated, leading to a significant increase of BrdU⁺/NeuN⁺ cells, indicating successful endogenous NSC proliferation and neuronal lineage differentiation. In the MT-AS-EVs group, a robust neurogenic response was observed compared with all other experimental groups. Taken together, our results indicated that melatonin-treated astrocytes can efficiently enhance neuronal expansion in a paracrine manner.

Fig. 6.

Monitoring endogenous proliferation and maturation in the ischemic mPFC region after administration of AS-EVs and MT-AS-EVs (A-B). The number of NeuN⁺/BrdU⁺ cells was assessed using IF at the lesion site after the injection of AS-EVs, and/or MT-AS-EVs into brain tissue. The data showed that MT-AS-EVs effectively increased the percentage of NeuN + and BrdU + cells at the injection sites compared with other groups. One-way ANOVA and Tukey post hoc analysis (n = 3). *p < 0.05; **p < 0.01; and ***p < 0.001

Trends in body weight of ischemic mice after MT-AS-EVs injection

Body weight was decreased in the sham group over the 21 days and may be attributed to ongoing neurological deficits, abnormal feeding behavior, and systemic stress (Fig. 7A). Data indicated that weight trajectories were more stable in IS mice receiving AS-EVs and MT-AS-EVs, with a greater impact in the MT-AS-EVs group. The injection of MT-AS-EVs led to a degree of systemic health and recovery, potentially facilitating not only nervous health but also the health of systems other than the nervous system following IS (Fig. 7A).

Fig. 7.

MT-AS-EVs promoted functional recovery and suppressed neuroinflammation in IS mice. Monitoring the body weight of experimental groups during the study indicated that both AS-EV and MT-AS-EV treatments resulted in more consistent weight trajectories (n = 10 per group), suggesting better overall health and recovery (A). ELISA tests showed a significant increase in pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine TGF-β in the sham group compared to the control, demonstrating post-ischemic neuroinflammatory responses. AS-EVs and MT-AS-EVs administration significantly reduced TNF-α and TGF-β levels when compared to the IS group, suggesting a robust anti-inflammatory effect of EV-based therapy (B-C; n = 3). Western blot analysis showed decreased expression of synaptic proteins (synaptophysin and Homer1a) in the IS group; treatment with AS-EVs demonstrated partial recovery, whereas MT-AS-EVs produced a more marked recovery. These results suggest that melatonin preconditioning may enhance AS-EVs’ synaptic restorative capabilities (D-G; n = Data originated from 6 pooled samples). One-way ANOVA and Tukey post hoc analysis. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001

Inflammatory cytokine profiles

ELISA results showed that TNF-α and TGF-β were at the highest levels in the sham group compared to the control mice (Fig. 7B-C; p < 0.05). The increase in these cytokines is likely related to an unmediated inflammatory response triggered by IS injury. The injection of MT-AS-EVs and AS-EVs reduced cytokine levels. It seems that the anti-inflammatory properties of AS-EVs were higher than MT-AS-EVs. Taken together, these data confirmed the potency of naïve and melatonin-treated astrocyte EVs in the restoration of immune cell function post-IS.

MT-AS-EVs promoted synaptogenesis in the ischemic mPFC region

Western blotting showed the changes of synaptic proteins between pre- and post-IS and MT-AS-EVs and AS-EVs groups (Fig. 7D-G). Induction of IS led to reduced synaptic protein levels in the sham group compared with control mice, indicating reduced synaptic maintenance due to untreated ischemia. Elevated expression of synaptophysin (a presynaptic vesicle marker) and Homer1 (a postsynaptic scaffolding protein) was observed in the MT-AS-EVs group, indicating upregulation of synaptic plasticity and structural repair. A different protein, Synapsin I, was also markedly elevated in the AS-EVs group compared to the MT-AS-EVs group. Synapsin I is also presynaptic, but it is linked to presynaptic vesicle trafficking, which indicates a partial presynaptic response to astrocyte-EV therapy (Fig. 7D-G).

Behavioral assessments and neuropsychiatric outcomes

To assess post-IS functional recovery and neuropsychiatric-like outcomes, a full panel of behavioral testing was conducted 21 days after the injection. These tests included the FWT and TST for depression-like behavior, the light/dark box test for anxiety-related behavior, and the OFT for overall locomotor activity plus anxiety control.

TST

Analogous trends were observed with the TST (Fig. 8A). In the sham group, high immobility, reflecting learned helplessness, was achieved. Treatment with MT-AS-EVs significantly reduced immobility time, suggesting that MT-AS-EVs have the potential to reverse affective dysfunction. In the AS-EVs group, a comparatively modest reduction in immobility was observed, suggesting limited but potentially present neurobehavioral benefits.

Fig. 8.

Behavioral analysis of IS mice after MT-AS-EVs and AS-EVs administration. Behavioral tests were conducted to evaluate depressive- and anxiety-like behaviors as well as locomotor and exploratory activity in all experimental groups (A-K). In TST (A) and FST (E), the IS group of mice exhibited significantly greater immobility times than control animals, indicative of behavioral changes typically associated with post-stroke depression. Treatment with AS-EVs reduced immobility time relative to sham mice, while MT-AS-EVs administration produced the most pronounced decrease, reflecting enhanced antidepressant-like effects. In the Light/Dark test, sham mice had a greater latency to enter the lighted chamber and spent a longer time in the dark, indicating anxiety-like behavior. MT-AS-EVs improved latency, increased time spent in the light zone, and increased the number of transitions, representing an anxiolytic effect (B-D). OFT data related to control (G), sham (H), AS-EVs (J), and MT-AS-EVs (K). The IS mice had limited mobility and spent time mostly in peripheral zones, which is indicative of anxiety and a decreased exploratory drive. By contrast, mice that received MT-AS-EVs showed a significant increase in locomotor activity and center zone entries, indicating improved motor recovery, reduced anxiety, and restored exploratory behavior

Light/dark box test

Anxiety-like behavior was assessed by measuring the latency to enter the light chamber, total time in each chamber, and the number of transitions (Fig. 8B-D). There was a delay in latency to enter the light zone for sham mice, the number of transitions was limited, and these mice spent significantly longer in the dark chamber, all indicative of anxiety-like behavior. In mice that received MT-AS-EVs, partially reversed parameters were achieved by spending considerably more time in the light chamber and demonstrating increased transitions into the light zone, indicating reduced anxiety-like behavior and improved exploration and behavioral flexibility. AS-EVs mice demonstrated intermediate values, suggesting partial anxiolytic properties. Overall, MT-AS-EVs exerted anxiolytic effects, presumably by reinstating a functioning neural circuit that had been conditioned to improve emotional regulation.

FST

The mice subjected to the FST showed a range of immobility times (Fig. 8E). The sham group showed significantly increased immobility, reflecting a high degree of behavioral despair and motivational reduction. The MT-AS-EV-treated mice showed a striking decrease in immobility time, suggesting a strong antidepressant-like effect likely via neuroprotection and recovery of synaptic processes. The AS-EVs group showed a reduction in immobility compared with the sham group, but to a lesser extent than the MT-AS-EVs group. Therefore, the MT-AS-EV therapy had significant effects on post-stroke depressive-like symptoms. This is consistent with the anti-inflammatory and synaptic remodeling effects observed in our biochemical assays.

OFT

Exploratory and anxiety-like behaviors were further examined by quantifying the distance traveled, time spent in the center compared to peripheral zones, and the number of center zone entries (Fig. 8F-K). The hypolocomotion observed in IS mice limits the number of center zone entries and the distance traveled. In contrast, resumed locomotor activity and increased entries into the center zone indicate that MT-AS-EV-treated mice have re-engaged in exploratory behavior, demonstrating both improved motor function and potentially reduced anxiety. Behaviorally, AS-EVs-treated mice showed intermediate levels of activity, indicating partial recovery. Treatment with MT-AS-EVs appeared to represent a clear and significant improvement in both the motor and emotional behavioral domains, as noted in the histological and molecular findings discussed, with evidence of synaptic and structural recovery. The decreased immobility in the depression-related tasks (FST and TST), in addition to normalized exploratory and anxiety-related behavior (Light/Dark Box and OFT), supports the idea that MT-AS-EVs provide the most effective neurobehavioral recovery in stroke. These neurobehavioral results closely correspond to molecular evidence of decreased neuroinflammation and increased synaptic protein expression in the MT-AS-EVs condition.

Discussion

Recent data have shown that EVs have potential in supporting the neuroregeneration of the injured CNS in various animal models [52]. Despite the existence of regenerative outcomes, most clinical trials have failed to achieve reliable regeneration in humans after the injection of EVs from different sources [53]. To enhance the regeneration properties of EVs after ischemic events, it will be necessary to implement strategies like choosing appropriately modified cell sources [54]. Here, we presented evidence that AS-EVs and MT-AS-EVs cause substantial neuroprotective, anti-inflammatory, and synaptogenic effects following photothrombotic IS in mice. The neuroprotective, anti-inflammatory, and synaptogenic effects were evaluated using multiple disciplines, including histopathology, metabolic profiling, and behavioral analysis. Melatonin conditioning improved astrocytic EVs compared to AS-EVs and IS controls.

Histological evaluations using Cresyl Violet and H&E staining showed greater cell loss and neuronal degeneration in the IS condition, consistent with the pathological stereotypical profile and characteristics of an IS. Treatment with AS-EVs yielded moderate tissue protection and limited cytoarchitectural preservation in the mPFC. In contrast, MT-AS-EV demonstrated a reduction in infarct volume compared to the sham condition and preserved cellular morphology, with histological evidence of tissue integrity. Prior studies have demonstrated the neuroprotective potential of EVs derived from different cell sources via multiple mechanisms. For example, plasma EVs pre-treated with melatonin yield a significant reduction in infarct cavity volume in rodent models of IS [55]. Therefore, the clinical implications of melatonin application are consistent with the broader body of evidence on its neuroprotective effects, which have been at least partially attributed to direct downstream improvements in cell survival and tissue integrity during IS [56]. Post-ischemic inflammation is a significant mediator of secondary brain injury. The ELISA assay showed elevated TNF-α and TGF-β levels in the IS group, indicating an inflammatory response. MT-AS-EV treatment reduced TNF-α levels, coincident with an increase in TGF-β levels, suggesting a possible shift from a pro-inflammatory to an anti-inflammatory state. This immunomodulatory response was consistent with melatonin’s immunoregulatory effects and the knockdown of neuroinflammation [57]. Similar responses were observed with EVs from hypoxia-preconditioned microglia, which elicited an anti-inflammatory M2-polarized response and improved outcomes after IS [58]. Western blot analysis showed significant increases in synaptic markers for presynaptic vesicle regeneration (synaptophysin) and postsynaptic structural recovery (Homer1). There was a slight increase in synapsin I expression with the AS-EVs, with minimal presynaptic recovery. These molecular changes showed improved synaptic plasticity and connectivity, as required for functional recovery post-stroke. The animals that received the MT-AS-EVs showed an increase in BrdU⁺/NeuN⁺ cells, indicating neurogenesis, or differentiation of neuron-like cells, particularly in the peri-lesioned cortex. The results are consistent with prior works reporting that melatonin and MT-AS-EVs enhance neurogenesis and synaptic remodeling following IS [59, 60]. Functional validation through behavioral assays at 21 days post-treatment showed that administration of MT-AS-EVs significantly improved depression-like (FST, TST) and anxiety-like behaviors (Light/Dark Box, OFT). The behavioral improvements are in line with molecular and histological recovery, indicating that MT-AS-EVs may help restore the cognitive and affective functions that IS impaired. While several exciting results were identified, it is important to recognize some limitations. First, we administered EVs as a single dose at a single time point, which does not accurately reflect the dosing regimens, schedules, or therapeutic window required for recovery. Moreover, there appears to be potential value in administering EVs in a repeated-dosing model, as examined in recent EV studies. Second, while increases in BrdU incorporation into new cells suggest neurogenesis, future studies will require lineage-tracing and functional integration studies to confirm that newly generated neurons are driving recovery. Third, it will be necessary to fully characterize the molecular cargo (miRNA and proteomic profiles) of the MT-AS-EVs, as this will help identify the mechanisms underlying enhanced efficacy. There will also be several logistical considerations for translating findings to human stroke patients: the choice of EV source, dosing and delivery methods, and the identification of potential long-term safety or efficacy concerns.

While existing studies have separately shown significant biocompatibility and negligible toxicity of both melatonin and astrocyte-derived EVs in a variety of experimental conditions [61, 62]. We did not include long-term assessment for potential adverse effects. The crucial next step before transitioning to clinical practice is analyzing safety, including biodistribution, immune activation, and potential off-target interactions. Future studies should include longitudinal assessments of the histology of peripheral organs and immunological evaluation of the systemic long-term safety of MT-AS-EV therapy.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AS-EVs

Astrocytes-Extracellular vesicles MT-AS-EVS

CNS

Central Nervous System

EVs

Extracellular vesicles

BBB

Blood-Brain Barrier

IF

Immunofluorescence

IS

Ischemic stroke

MCAO

Middle Cerebral Artery Occlusion

MAEV

Melatonin-Astrocytes-Extracellular vesicles

mPFC

Medial prefrontal cortex

PFC

Prefrontal cortex

PSD

Post-stroke depression

r-tPA

Recombinant tissue plasminogen activator

Author contributions

Behnaz Mirzaahmadi conducted all experiments and tests, contributed to conceptualization, data curation, and wrote the original draft. Dr. Hajar Shafaie and Dr. Javad Mahmoodi supervised the experimental work. Parinaz Haddadi and Ali Hassanzadeh assisted in the experimental procedures. Prof. Russel J. Reiter provided scientific consultation, critical review, and final approval of the manuscript. Dr. Mohammad Karimipour and Prof. Reza Rahbarghazi supervised the overall project and contributed to critical revision and manuscript editing. All authors read and approved the final version of the manuscript.

Funding

This study was supported by a grant (No: 71246) from Tabriz University of Medical Sciences and the Vice President of Scientific Technology and Knowledge Council for the Development of Regenerative Medicine and Stem Cells Technologies (No: 11-121522). The funding sources had no role in the design of the study, in the collection, analysis, and interpretation of data, or in writing this manuscript or deciding to submit it for publication.

Data availability

Data will be available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was registered as titled “The evaluation of the effect of melatonin-pretreated astrocyte-derived exosomes on neurogenesis and neural tissue regeneration in the medial”. All steps of this study were approved by the Ethical Committee of Tabriz University of Medical Sciences under the ethical code of (IR.TBZMED.AEC.1402.020) on 2023-05-29. Animals were treated according to the previously published ARRIVE guidelines.

Consent for publication

Not applicable.

Conflict of interest

None declared.