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Abstract

Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have shown promise in promoting functional recovery in male rodent models of traumatic brain injury (TBI). This study aimed to assess the effects of human MSC-derived sEVs on functional recovery, neuroprotection, and neuroinflammation in female rats subjected to TBI. Young female rats were subjected to TBI induced by controlled cortical impact injury over the left parietal cortex. sEVs or phosphate-buffered saline (PBS) was administered intravenously 1 day post-injury. A battery of sensorimotor and cognitive functions was assessed post-injury. At day 35, brains were collected for histological analyses of lesion volume, neuroprotection, and neuroinflammation. Compared to the PBS treatment, sEVs significantly reduced the frequency of foot-faults, adhesive removal time, and modified Neurological Severity Scores (p < 0.05 vs. PBS). Moreover, sEVs significantly enhanced cognitive abilities, demonstrated by a shorter time to locate the hidden platform and an increased percentage of time spent in the target quadrant during the Morris water maze test (p < 0.05). Furthermore, sEV administration significantly decreased the size of brain lesions, reduced hippocampal neuronal cell loss, increased synaptophysin expression, and attenuated neuroinflammation following injury (p < 0.05). These findings, in conjunction with previous studies of sEV benefits in male TBI rats, indicate that delayed (1 day post-injury) intravenous delivery of human MSC-derived sEVs could be an effective treatment approach for TBI in both sexes.

Keywords

females functional outcome neuroprotection neuroinflammation rat small extracellular vesicles traumatic brain injury

Introduction

Traumatic brain injury (TBI) is a leading contributor to lasting disabilities across the globe, with limited therapeutic options available to promote brain healing and functional restoration of the damaged brain.^1–3 The pathophysiology of TBI involves multiple processes, including neuroinflammation, neuronal cell death, and the blood–brain barrier (BBB) breakdown, which result in functional deficits that may continue well beyond the initial trauma.^1,2 Although significant progress has been made in uncovering the molecular processes involved in TBI, currently, there are no successful clinical therapies that ensure neuroprotection or restore neurological function effectively.⁴ In recent years, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have gained attention as a promising cell-free treatment option for TBI, owing to their capacity to transport bioactive molecules that can modulate inflammation, enhance tissue repair, and promote neuroprotection.^5,6

MSC-derived EVs, which include small EVs (sEVs) also termed as exosomes, contain bioactive molecules, including proteins, lipids, DNA, RNA, and microRNAs (miRNAs), that can regulate cellular processes of recipient cells and promote healing in the injured brain.^7,8 These vesicles have demonstrated the ability to modulate inflammation, a key driver of secondary brain injury in TBI.^5,7–9 For instance, sEVs from MSCs have been found to suppress the activation of microglia and astrocytes—major drivers of neuroinflammation—leading to decreased production of pro-inflammatory cytokines and fostering conditions that support tissue healing.^9–11 Furthermore, MSC-derived sEVs can stimulate neurogenesis, enhance neuronal survival, and improve synaptic plasticity, all of which are crucial for functional recovery after TBI.^12,13

The advantages of using MSC-derived sEVs over live MSCs are numerous. First, sEVs are more stable and easier to administer, as they can be delivered via systemic routes such as intravenous injection without the complications associated with stem cell transplantation including vascular occlusion.^14,15 Additionally, sEVs do not pose the same risks as live cells, including tumor formation or immune rejection, making them a safer alternative for clinical use.^14,15 Moreover, MSC-derived sEVs can cross the BBB.¹⁶ This ability to penetrate the BBB enables targeted therapeutic effects on the injured brain tissue, a key limitation in many other drug and cell treatment strategies.^17,18

Despite the growing interest in using sEVs as a therapeutic strategy for TBI, most pre-clinical studies have predominantly focused on male subjects,^9,19 and there remains a significant gap in research involving female animal models. This lack of investigation into female TBI models not only limits our understanding of the efficacy and safety of sEVs across sexes but also hinders the development of inclusive and effective therapies. Given the known differences in TBI pathophysiology and recovery between males and females documented,^20,21 it is critical to explore the therapeutic role of MSC-derived sEVs in female TBI models to thoroughly evaluate their therapeutic potential for wider clinical use. This research for the first time evaluated the therapeutic effects of administering MSC-derived sEVs intravenously 1 day after injury on enhancing functional recovery, promoting neuroprotection, and inhibiting neuroinflammation in a female rat model of TBI.

Methods

Isolation and analyses of human MSC-sEVs

The culture of human MSCs (hMSCs) and the isolation of sEVs were carried out following our previously published protocol.⁹ hMSCs, supplied by Theradigm (Bethesda, MD), were expanded as described in earlier studies.^22,23 Briefly, passage 5 hMSCs were seeded at a density of 3 × 10⁶ cells per 75 cm² flask using low-glucose Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY), supplemented with 20% fetal bovine serum (Gibco BRL), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L l-glutamine. Upon reaching 60–80% confluency, the regular culture medium was replaced with exosome/sEVs-depleted fetal bovine serum medium (EXO-FBS-250 A-1; System Biosciences, Mountain View, CA), and cells were cultured for an additional 48 h. The conditioned medium was then collected, filtered through a 0.22 μm filter (Millipore, CA), and centrifuged at 10,000 g for 30 min. The resulting supernatant was subjected to ultracentrifugation at 100,000 g for 3 h to pellet the sEVs/exosomes. The pellet was resuspended in sterile phosphate-buffered saline (PBS) for subsequent characterization and experiments. The particle concentration and size distribution of the sEVs-enriched pellet were determined using nanoparticle tracking analysis (NTA) with a NanoSight instrument (Merkel) and validated by transmission electron microscopy (TEM) (JEOL JEM-1400Flash), as we previously described.²⁴ Protein concentration of the sEVs was measured using the bicinchoninic acid assay kit (Pierce, Rockford, IL). Finally, Western blotting was performed to confirm the presence of exosomal/sEV markers CD63 (1:1000, Santa Cruz, sc-5275), CD9 (1:500, Abcam, ab223052), Alix (3A9) (1:1000, Cell Signaling, 2171), and exosome/sEV negative marker calnexin (1:1000, Biolegend, 699401) in the isolated sEVs according to our previous method.²⁵ All these characterizations meet minimal information for studies of EVs (MISEV) guidelines.^26,27

Experimental TBI model

A controlled cortical impact (CCI) model was utilized to induce TBI,²⁸ days following the methodology detailed in our earlier work.⁹ Briefly, naturally estrous-cycling female Wistar rats (aged 2–3 months), purchased from Charles River Laboratories (Wilmington, MA), were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and then secured in a stereotaxic apparatus. An 8 mm craniotomy was made over the left parietal cortex without disrupting the dura mater. A moderate TBI was then delivered using a pneumatic impactor with a 6 mm tip, traveling at a velocity of 4 m/s and penetrating to a depth of 2.5 mm, in accordance with our previously reported protocol.⁹

Treatment assignments

TBI rats were randomly assigned to receive one of the following treatments: (1) PBS (Vehicle group, n = 8) or (2) sEVs derived from human MSCs (sEVs group, n = 8). One day after injury, rats were given an intravenous injection via the tail vein of either sEVs (100 µg protein, 3.75 × 10¹¹ particles in 0.5 mL PBS per rat) or Vehicle (0.5 ml PBS) as a treatment control. Sham rats underwent the same surgical procedure without TBI or treatment and served as the control group (Sham group). The sEV dosage and timing were determined based on our previous research involving exosome/sEV therapy for TBI.⁹ The 100 μg total protein of exosomes/sEVs injected into each rat was collected from approximately 2 × 10⁶ MSCs, a number of MSCs equivalent to the effective amount that we previously used in the MSC-based treatment for TBI (2 × 10⁶ MSCs per rat).²⁹

Functional outcome tests

Sensorimotor assessments, such as foot-fault, adhesive removal, and the modified Neurological Severity Score (mNSS), were conducted 1 day after injury and then weekly for 5 weeks. Spatial learning and memory were evaluated using the MWM test between days 31 and 35 post-injury.³⁰ Detailed methods for these established tests are outlined in our previously published protocol.⁹

mNSS test

Neurological function was assessed using the mNSS test. Evaluations were conducted on all rats on days 1, 7, 14, 21, 28, and 35 after TBI. The mNSS test combines motor function (such as muscle tone and abnormal movements), sensory responses (including visual, tactile, and proprioceptive), and reflexes.⁹ Scores range from 0 to 18, with 0 representing normal function and 18 indicating the most severe impairment. Each observed deficit or missing reflex adds one point, meaning higher scores reflect greater neurological damage.

Foot-fault test

To assess sensorimotor abilities, the foot-fault test was performed on days 1, 7, 14, 21, 28, and 35 following injuries. During the test, rats walked across a wire grid, and any time the right forelimb slipped or missed a step through the gaps, it was counted as a foot fault.⁹ A total of 100 steps were monitored for the right front paw.

Adhesive removal test

In short, two adhesive-backed paper dots (113.1 mm² each) were placed on the distal-radial area of each forelimb to serve as tactile stimuli. Each rat underwent three trials per test day, and the average time (in seconds) it took to remove the adhesive from the right forelimb was recorded. This average was used for statistical analysis. The test was conducted on days 1, 7, 14, 21, 28, and 35 post-TBI.

Morris water maze test

To assess spatial learning and memory deficits, a modified version of the Morris water maze (MWM) test was adapted from earlier protocols³⁰ and applied in brain injury models.^9,12 Testing occurred on days 31–35 post-TBI, with animals completing four trials per day over 5 days. The setup involved a 1.8 m diameter blue pool located in a room with fixed visual cues (e.g., wall posters, lamps, ceiling camera) for spatial orientation. Each trial began with the rat placed at one of four preset starting points (North, South, East, or West), facing the wall. Rats had 90 sec to locate a hidden, transparent platform placed 2 cm below the water in the northeast (NE) quadrant. If successful, they remained on the platform for 10 sec; otherwise, they were guided to it for 10 sec. The platform’s position varied within the NE quadrant—against the wall, near the center, or off-center—requiring rats to rely on external cues for navigation.

The pool was divided into four quadrants for data tracking using the HVS Image 2020 Plus System (US HVS Image, San Diego, CA). Time spent in the NE quadrant before reaching the platform and escape latency were recorded for statistical analysis. Unlike the traditional MWM, where the platform remains fixed, this version randomly repositions the platform within the target quadrant during each trial. This design forces rats to rely more heavily on spatial cues, making every trial similar to a probe trial and enhancing the accuracy of cognitive assessment.³⁰

Histological analyses

At 35 days after injury, rats were anesthetized using ketamine/xylazine and underwent transcardial perfusion with saline, followed by 4% paraformaldehyde. The brains were then collected and fixed in 4% paraformaldehyde for 2 days. Brain tissues were embedded in paraffin, sectioned into 6-μm-thick slices from 2-mm blocks, and stained using hematoxylin and eosin.⁹ Lesion volumes (mm³) were calculated by multiplying the measured area (mm²) of each brain section by the distance (mm) between sections. The results were then expressed as a percentage relative to the uninjured right hemisphere.⁹

Immunohistochemistry

Briefly, antigen retrieval was achieved by boiling brain sections in 10 mM citrate buffer (pH 6.0) for 10 min. After rinsing three times with PBS, sections were treated with 0.3% hydrogen peroxide in PBS for 10 min and then blocked with 1% bovine serum albumin (BSA) containing 0.3% Triton X-100 at room temperature for 1 h. Sections were incubated overnight at 4°C with primary antibodies: anti-neuronal nuclei (NeuN) (1:300, MAB 377, Chemicon), anti-CD68 (1:200, Serotec, UK), mouse anti-synaptophysin IgG1κ, clone SY38 (1:500, Millipore, MAB5258-50UG), or anti-glial fibrillary acidic protein (GFAP) (1:1000, Dako, Denmark). Negative controls were processed without primary antibodies. Following incubation, sections were rinsed and treated with biotinylated anti-mouse or anti-rabbit secondary antibodies (1:200, Vector Laboratories) for 30 min at room temperature. After further washing, an avidin–biotin–peroxidase complex (ABC kit, Vector Laboratories) was applied. Visualization was done using diaminobenzidine (Sigma), and sections were counterstained with hematoxylin.

Cell counting and quantitation

Cluster of differentiation (CD68)+ microglia/macrophages, NeuN+ neurons, synaptophysin+ areas, and GFAP+ astrocytes were quantified in the injured cortex and hippocampus. In the present study, we did not use stereological quantification methods, as immunoreactive positive cells are not homogenously distributed³¹ and the data of interest are relative differences and not absolute values.^32,33 For quantitative measurements of immunopositive cells, every sixth coronal section for a total of five sections with 40 μm intervals of the brain containing the hippocampus with coordinates of bregma from −3.14 to −4.30 mm was used for immunohistochemical staining and analyzed with a microscope (Nikon i80) at 200× or 400× magnification via the MCID system. Briefly, five fields of view in the lesion boundary zone from the epicenter of the injury cavity (Bregma −3.3 mm) and nine fields of view in the ipsilateral dentate gyrus (DG) were counted in each section. All counting was performed on a computer monitor to improve visualization and in one focal plane to avoid oversampling.³⁴ This method permits a meaningful comparison of differences among groups. The fields were digitized under the light microscope (Nikon, Eclipse 80i, Melville, NY) at either 200× or 400× magnification using a CoolSNAP color camera (Photometrics, Tucson, AZ) interfaced with MetaMorph image analysis system (Molecular Devices, Downingtown, PA). The immunopositive cells or areas were measured and presented as numbers per mm² or % area. Cell counts and area measurements were performed by observers blinded to the individual treatment status of the animals. To evaluate whether sEVs reduce neuronal damage after TBI, the number of NeuN+ neuronal cells was counted in the DG, CA1, and CA3 regions. Cell counts were averaged and normalized by measuring the area (in mm²) of these regions for each section.

Statistical analyses

All results were presented as mean ± standard deviation. Statistical differences in histology were assessed using analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons. Repeated-measures ANOVA, followed by Tukey’s post hoc test, was used to evaluate spatial learning and sensorimotor performance over time. A p value <0.05 was regarded as statistically significant.

Results

Identification of MSC-sEVs

sEVs were characterized by (1) NTA reports full particle-size distributions and concentrations (yield) (Supplementary Fig. S1); (2) TEM demonstrates canonical vesicle morphology with an enlarged inset (Supplementary Fig. S1B); and (3) full-length, uncropped Western blots show the EV markers CD63, CD9, Alix and the absence of calnexin (non-EV/endoplasmic reticulum marker), supporting preparation purity (Supplemental Fig. S1C). Together, these data document size, morphology, marker profile, yield, and purity in line with current MISEV2018/2023 recommendations.^26,27

MSC-sEV treatment significantly improves spatial learning and memory in female rats following TBI

Spatial learning and memory were evaluated using a modified MWM test, a method particularly sensitive to hippocampal damage and associated cognitive impairments.³⁰ Spending a greater proportion of time in the target quadrant (NE, where the hidden platform was located) reflects improved spatial memory. Sham-operated rats demonstrated a significant increase in time spent in the correct quadrant and a decrease in the time required to locate the platform between days 32 and 35 compared to day 31 (Fig. 1). Rats receiving sEV treatment spent significantly more time in the target quadrant (Fig. 1A, p < 0.05) and demonstrated a considerable decrease in the time taken to locate the platform between days 33 and 35 compared to the vehicle group (Fig. 1B, p < 0.05).

FIG. 1.

MSC-sEV treatment markedly improves spatial learning after TBI, as shown by the increased percentage of time animals spent in the target quadrant with the hidden platform (A) and the reduced time required to find the platform (B). Values are shown as mean ± standard deviation, with eight animals in each group. MSC, mesenchymal stem cell; sEV, small extracellular vesicles; TBI, traumatic brain injury.

MSC-sEV treatment significantly enhances sensorimotor recovery in female rats following TBI

Sensorimotor abilities in rats were evaluated using the mNSS test. One day following TBI, both the PBS and sEV-treated groups showed similar mNSS scores, indicating equivalent levels of neurological impairment before treatment (Fig. 2A, p > 0.05). Over time, PBS-treated rats showed a gradual improvement in function, with significantly lower mNSS scores from days 7 to 35 compared to day 1, reflecting natural recovery. However, rats that received sEVs showed a significantly greater reduction in mNSS scores than the PBS group during the same period (p < 0.05). Moreover, sEV treatment resulted in a notable reduction in right forelimb foot-faults (Fig. 2B, p < 0.05) and shortened adhesive removal times from days 7 to 35 compared to PBS-treated controls (Fig. 2C, p < 0.05).

FIG. 2.

MSC-sEV treatment significantly enhances neurological recovery following TBI, measured by mNSS (A), foot-fault test (B), and adhesive removal test (C). Results are expressed as mean ± standard deviation, with eight rats per group. mNSS, modified Neurological Severity Score; MSC, mesenchymal stem cell; sEV, small extracellular vesicles; TBI, traumatic brain injury.

MSC-sEV administration significantly reduces brain inflammation in female rats after TBI

CD68 staining was used to detect macrophages and microglia in the brain on day 35 following TBI. Compared to sham controls, TBI alone caused a significant rise in the number of CD68+ cells in the lesion boundary zone (p < 0.05) and DG (p < 0.05) of the ipsilateral hemisphere (Fig. 3, left panel). Treatment with sEVs significantly decreased the density of CD68+ cells in both the injured cortex and DG compared to PBS-treated animals (Fig. 3, left panel, p < 0.05).

FIG. 3.

MSC-sEV treatment leads to a significant decrease in the expression of CD68+ microglia/macrophages and in the GFAP+ astrocytes observed 35 days post-TBI. Left panel: CD68 staining in the cortex (A–C) and DG (D–F) of the ipsilateral brain. Right panel: GFAP staining in the cortex (A–C) and DG (D–F) of the ipsilateral brain. Scale bar = 100 µm. Quantitative data shown in bar graphs (G in the left and right panels) represent mean ± standard deviation. N = 8/group. DG, dentate gyrus; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline; sEV, small extracellular vesicles; TBI, traumatic brain injury.

GFAP staining was conducted to detect reactive astrocytes in the brain post-TBI. The TBI group showed a significant increase in GFAP+ cell density within the injured cortex and DG compared to sham controls (Fig. 3, right panel, p < 0.05). In contrast, sEV-treated rats exhibited a marked reduction in GFAP+ astrocyte density in the affected cortex and DG relative to those treated with PBS (Fig. 3, right panel, p < 0.05).

MSC-sEV administration significantly reduces cortical lesion volume and neuronal cell loss in female rats after TBI

All of the animals survived the 35-day study. Cortical lesion volume was evaluated 35 days post-TBI, as described previously,⁹ and was significantly smaller in the sEV-treated group compared to the PBS group (Fig. 4, p < 0.05). Neuronal staining was used to identify mature neurons at 35 days post-injury. TBI led to a significant reduction in the number of NeuN+ neurons in the CA1, CA3, and DG areas of the damaged hippocampus (Fig. 5, p < 0.05 vs. Sham). However, sEV treatment offered significant protection against neuronal loss in these regions compared to PBS-treated rats (Fig. 5, p < 0.05 vs. PBS).

FIG. 4.

Treatment with MSC-sEVs significantly reduces cortical lesion volume 35 days after TBI (hematoxylin and eosin staining). Scale bar = 2 mm. Quantitative data shown in bar graphs represent mean ± standard deviation. N = 8/group. MSC, mesenchymal stem cell; PBS, phosphate-buffered saline; sEV, small extracellular vesicles; TBI, traumatic brain injury.

FIG. 5.

sEV-treatment markedly lessens neuronal loss in the ipsilateral hippocampus following TBI. NeuN staining in the DG (A, D, G), CA1 (B, E, H), and CA3 (C, F, I) regions. Scale bar (I) = 100 µm. Quantitative data shown in bar graphs (J) represent mean ± standard deviation. N = 8/group. DG, dentate gyrus; PBS, phosphate-buffered saline; sEV, small extracellular vesicles; TBI, traumatic brain injury.

MSC-sEV administration significantly reduces synaptophysin loss in female rats after TBI

We performed synaptophysin immunostaining to evaluate the effects of sEVs on synaptogenesis. TBI led to a significant reduction in synaptophysin immunoreactivity in the injured cortex and hippocampus (Fig. 6, p < 0.05 vs. Sham). However, sEV treatment significantly attenuated loss of synaptophysin immunoreactivity compared to PBS-treated rats (Fig. 6, p < 0.05 vs. PBS).

FIG. 6.

Treatment with MSC-sEVs significantly reduces synaptophysin loss after TBI. Synaptophysin staining in the ipsilateral cortex and DG of Sham (A, D), PBS (B, E), and sEVs groups (C, F). Scale bar (F) = 50 µm. Quantitative data shown in bar graphs (G) represent mean ± standard deviation. N = 8/group. DG, dentate gyrus; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline; sEV, small extracellular vesicles; TBI, traumatic brain injury.

Discussion

Although sEVs have been investigated as a pre-clinical therapeutic strategy for TBI,^5,24 a weakness is that its efficacy is not well established in female rats with TBI. This weakness is critical because females make up a considerable proportion of TBI survivors who require effective therapies. It is important to assess the potential efficacy of sEVs in promoting sensorimotor and cognitive enhancement as well as reducing brain damage and neuroinflammation in female rats after TBI using the same injury model and sEVs treatment paradigm (i.e., initiation and duration) as previously reported.⁹ This study aimed to validate the beneficial effects of MSC-sEVs on functional recovery and histology in female TBI rats. Therefore, we followed experimental design, functional assays, immunostaining markers, and end-points largely identical to our previous work in male rats.⁹ Some of the previous reports revealed that the estrous stage at the time of TBI did not impact subsequent behavior,^35–38 suggesting that the presence of endogenous circulating hormones, rather than hormonal status at the time of injury, may confer early neuroprotection in females after TBI.³⁵ However, other reports indicate the estrous cycle significantly influences TBI outcomes in rats, and variations in female sex hormone levels may explain the differing degrees of neuroprotection observed between proestrus and nonproestrus groups.³⁹ In this study, we chose to injure naturally estrous-cycling female rats due to the clinical applicability (i.e., real-world experiences).

The results of this study demonstrate that a single moderate CCI-TBI produces significant and lasting effects on neurological function in female rats, including both cognitive and sensorimotor impairments up to 35 days post-injury. These deficits are linked to neuronal and synaptophysin loss and elevated neuroinflammation in the injured brain. Moreover, a single dose of delayed (1 day post-injury) intravenous administration of human MSC-derived sEVs significantly enhances functional and histological recovery in female rats following TBI. These findings are particularly important considering the long-standing underrepresentation of females in pre-clinical TBI studies and the increasing recognition of sex-based differences in TBI progression and treatment outcomes.^20,21,40,41sEV treatment in female rats led to improvements in sensorimotor function, as evidenced by reductions in foot-fault errors and adhesive removal latency. Importantly, while female rats displayed some degree of spontaneous recovery, the vehicle-treated group continued to exhibit noticeable impairments up to 35 days after the injury. This underscores the need for interventions that can augment or accelerate recovery beyond natural compensatory mechanisms. sEV-treated animals also exhibited significant improvements in neurological outcomes as measured by the mNSS, which integrates sensory, motor, and reflex tests.⁹ These improvements suggest that sEVs exert a broad neuroprotective effect across multiple functional domains. Evaluation of cognitive performance using the MWM showed significant improvement in spatial learning and memory in the group treated with sEVs. These findings are translationally relevant, as cognitive impairments are among the most debilitating long-term consequences of TBI.⁴² The decreased escape latency and greater duration spent in the target quadrant observed in sEV-treated animals indicate preserved or restored hippocampal function.

Furthermore, the treatment significantly attenuated neuroinflammatory responses, as demonstrated by the reduced presence of CD68+ microglia/macrophages and GFAP+ astrocytes in the injured brain. CD68 staining revealed a substantial increase in activated macrophages and microglia within the lesion boundary zone of the injured cortex and DG on Day 35 post-TBI, indicating sustained inflammation in the injured brain. However, treatment with sEVs markedly reduced the number of CD68+ cells in both regions compared to PBS-treated animals, suggesting that sEVs effectively suppress microglial/macrophage activation. Similarly, GFAP staining showed elevated levels of reactive astrocytes in the injured cortex and DG of TBI animals, further confirming glial activation as a hallmark of secondary injury. Notably, sEV treatment significantly reduced GFAP+ astrocyte density, demonstrating its anti-inflammatory effect on astroglial reactivity. Together, these findings suggest that sEVs can attenuate chronic neuroinflammation as evidenced by reducing microglia/macrophages and glial activation after TBI, underscoring their potential as a therapeutic strategy to improve long-term outcomes. These results also imply that sEVs may modulate the neuroinflammatory milieu post-injury, potentially creating a more permissive environment for tissue repair and plasticity.

sEV treatment provided neuroprotective effects, demonstrated by a decrease in lesion size. Moreover, our data demonstrate that MSC-derived sEVs reduced neuronal cell loss in the DG, CA1, and CA3 regions of the hippocampus, indicating that MSC-derived sEVs provide neuroprotection in these hippocampal regions. The neuroprotective effect of sEVs on the hippocampus may contribute to improved cognitive functional recovery because the hippocampus plays a key role in learning and memory.⁴³ Our data show that TBI reduced synaptophysin immunoreactivity in the injured cortex and hippocampus, while sEV treatment significantly attenuated the loss of synaptophysin immunoreactivity. Synaptophysin is a crucial protein located in presynaptic vesicles, and its levels are considered a reliable marker for axonal integrity and synapse density.^44,45 TBI can cause damage to neurons and lead to the loss of synapses and disruption of synaptic plasticity, which is linked to long-term behavioral disorders.^45–49 Some studies show that synaptophysin expression is decreased after TBI, reflecting the synaptic damage.^50,51 However, another study demonstrates an increase in synaptophysin expression after TBI, which might be a compensatory response.⁵²

The beneficial effects of MSC-derived sEVs on functional recovery and histology in female animals have not been investigated after an isolated TBI. An isolated TBI is a brain injury occurred without any other injuries, such as chest, abdomen, spinal, limb injury or hemorrhagic shock. Our present study is particularly significant considering the historical underrepresentation of female subjects in TBI research and highlighting the potential of sEVs as a sex-independent, minimally invasive therapeutic strategy. The therapeutic benefits observed in female TBI rats in the present study agree with those previously reported in male TBI rats,⁹ suggesting that sEVs offer a sex-independent approach to TBI treatment. This is particularly noteworthy given emerging data on sex-specific responses to brain injury and treatments.^20,21,41,53 Recent studies show that sEV/exosome treatment reduces the severity of neurological damage and promotes quicker neurological recovery in a clinically relevant large animal model (female swine) of severe TBI and hemorrhagic shock by offering neuroprotection and enhancing BBB integrity.⁵⁴ Our present study, along with the previous research, offers novel evidence that MSC-derived sEVs represent a promising and minimally invasive treatment option for TBI applicable to both males and females.

The current study has several limitations. First, while we assessed multiple functional and histological outcomes, the precise molecular mechanisms underlying sEV-mediated recovery remain to be elucidated. Future studies will explore the cargo composition of sEVs and their specific targets in the injured brain. Second, further research will determine the dose response, therapeutic window, and duration of sEVs in different models of TBI in both sexes. Third, longer-term studies are needed to determine the durability of the observed benefits. Fourth, our previous work focused on the beneficial effects of human MSC-derived exosomes/sEVs in male rats after TBI.⁹ This study aimed to validate the beneficial effects of MSC-derived sEVs on functional recovery and histology in female TBI rats. Our present study is significant considering the historical under-representation of female subjects in TBI research and highlights the potential of sEVs as a sex-independent, minimally invasive therapeutic strategy. Further hypothesis-driven studies need to focus on new mechanistic insight of sEVs in female TBI rats. Our previous work on analyzing the miRNA content within MSC-derived sEVs/exosomes,^24,55 engineering human MSCs to generate sEVs/exosomes with overexpression⁵⁵ and reduction²⁴ of miRNA levels, and evaluating the role of miRNAs in male rats after TBI underscores the important role of miRNAs in MSC-derived sEVs in the treatment of TBI by providing neuroprotection and anti-inflammation in male rats.^24,55 Sixth, we used manual profile cell counting in this study. Unbiased stereology is an ideal method for quantification applied to structures with a homogeneous distribution of cell bodies.⁵⁶ Previous studies indicate that profile counting methodology should be chosen when cell populations are heterogeneously distributed.⁵⁷ Some studies show that profile counts and stereological estimates result in identical results.⁵⁸

Conclusion

This study provides convincing evidence that a single moderate TBI leads to sustained cognitive and sensorimotor impairments in female rats, associated with cortical lesion, neuronal loss, synaptophysin reduction, and heightened neuroinflammation. Notably, delayed (1 day post-injury) intravenous administration of human MSC-derived sEVs significantly improved both functional outcomes and brain tissue integrity. The findings from our earlier work and present study support that MSC-sEVs hold potential as a novel, neuroprotective, sex-independent, minimally invasive therapeutic strategy. Future work is warranted to focus on optimizing treatment protocols (treatment dose, window, and duration), exploring the effects of MSC-sEVs acting on other brain cells and underlying mechanisms, evaluating long-term effects in multiple TBI models and different ages of both sexes to support clinical translation across diverse populations.

Transparency, Rigor, and Reproducibility Summary

The animal use protocol was approved by the Institutional Animal Care and Use Committee at Henry Ford Health, and the experiments in this study were conducted following the recommendations in the Guide for the Care and Use of Laboratory Animals. The rats were maintained under controlled environmental conditions including a standard 12-h light–dark cycle with a stable temperature and relative humidity, free access to food and water throughout the study. We implemented a rigorous experimental design in this pre-clinical study, following the methods outlined in our earlier animal research.⁵⁹ Animals with TBI were randomly allocated to receive either sEV treatment or a vehicle control via tail vein 24 h post-injury. Researchers responsible for functional evaluations and histological assessments were blinded to group assignments. The study involved 24 female Wistar rats, with no mortality or exclusions from the data analysis. Three groups of animals (Sham, PBS, sEVs) were used to produce the results shown in Figures 1–6 (N = 8 per group). The CCI model is a well-established TBI model that is widely used in TBI research. The outcome measures used (functional tests and histology) are well established both in the field and in our laboratory. Each behavioral and immunohistochemistry experiment included eight animals per group, with group sizes determined based on prior experience with the assays and an a priori power analysis using estimated effect sizes. Behavioral assessments (including sensorimotor and Morris water maze tests) and histological data were analyzed for normality and equal variance before conducting statistical comparisons and using repeated-measures ANOVA, followed by Tukey’s post hoc test. No external replication or validation studies have been performed or planned at this time of writing. The authors agree to provide the full content of the article on request by contacting the corresponding author.

Authors’ Contributions

Yanlu Z. and Yi Z.: Data acquisition, data analysis, interpretation, and article critical review. M.C. and Z.G.Z.: Conception and design, and article critical review. H.P.: Data acquisition. L.C.: Data acquisition and article critical review. A.M.: Article critical review. Y.X.: Conception and design, data acquisition, data analysis and interpretation, article drafting, and article critical review. All authors have reviewed and approved the final article for submission.

Footnotes

Acknowledgment

The authors thank Qinge Lu for technical assistance in histology.

Author Disclosure Statement

The authors declare no conflicts of interest.

Funding Information

This study was supported by National Institutes of Health/National Institute of Neurological Disorders and Stroke grant R01NS100710 (to Y.X.).

Data Availability

The original contributions presented in this study are included in the article material. Further inquiries can be directed to the corresponding author.

Supplemental Material

Abbreviations

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Human Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Attenuate Brain Damage and Improve Functional Recovery in Female Rats after Traumatic Brain Injury

Abstract

Keywords

Introduction

Methods

Isolation and analyses of human MSC-sEVs

Experimental TBI model

Treatment assignments

Functional outcome tests

mNSS test

Foot-fault test

Adhesive removal test

Morris water maze test

Histological analyses

Immunohistochemistry

Cell counting and quantitation

Statistical analyses

Results

Identification of MSC-sEVs

MSC-sEV treatment significantly improves spatial learning and memory in female rats following TBI

MSC-sEV treatment significantly enhances sensorimotor recovery in female rats following TBI

MSC-sEV administration significantly reduces brain inflammation in female rats after TBI

MSC-sEV administration significantly reduces cortical lesion volume and neuronal cell loss in female rats after TBI

MSC-sEV administration significantly reduces synaptophysin loss in female rats after TBI

Discussion

Conclusion

Transparency, Rigor, and Reproducibility Summary

Authors’ Contributions

Footnotes

Acknowledgment

Author Disclosure Statement

Funding Information

Data Availability

Supplemental Material

Abbreviations

References