Two-month-old male C57BL/6J mice procured from Jackson Laboratories (Bar Harbor, ME, USA) were utilized. The Animal Care and Use Committee of Texas A&M University approved all procedures performed on animals. A schematic diagram showing the timelines of experiments and analyses performed in this study is illustrated in Figure 1. Animals were randomly assigned to naïve control, sham surgery, and TBI groups (n = 10/group). Ninety minutes after the induction of controlled cortical impact injury (CCI), animals in TBI groups were treated with an IN dose of vehicle (phosphate buffered saline [PBS]; referred to as TBI) or hMSC-EVs (6.4, 12.8, or 25.6 × 10⁹ EVs/mice) (Kodali et al., 2023). A subgroup of animals in every group (n = 6/group) received intraperitoneal injections of 5′-bromodeoxyuridine (BrdU, 100 mg/Kg/day for 7 days) at post-TBI days 57–63 after sham or TBI surgery for neurogenesis analysis. Age-matched subgroups of animals in the naïve control and sham surgery groups neither received vehicle nor EVs. However, they were subjected to similar BrdU injections at time points matching the TBI groups. Eighty-four days post-TBI (equivalent to 3 weeks post-BrdU injections), animals receiving BrdU in all groups were perfused intracardially with 4% paraformaldehyde and brain tissues were processed for immunohistochemical and immunofluorescence studies. Fresh brain tissues were harvested from a second subgroup of animals in every group (n = 4/group) for biochemical assays. The age of the mice at the time of euthanasia was ~5 months in these long-term studies. Furthermore, to investigate changes in the concentration of mature BDNF, p-ERK1/2, and p-CREB in the hippocampus ipsilateral to CCI, additional subgroups of naïve control mice (n = 6), TBI mice receiving the vehicle (n = 6), and TBI mice receiving 25.6 × 10⁹ EVs (n = 6) were euthanized at 48 h post-treatment, and fresh brain tissues were harvested. The various treatment groups and the number of mice employed per group for different data collection and analyses are presented in Table 1. The animal groups and treatments were blinded to investigators who collected and analyzed data.

A unilateral CCI was induced in animals belonging to TBI and TBI + hMSC-EV groups using a CCI device (Leica Biosystems, Deer Park, IL, USA) attached to stereotaxic equipment (MyNeurolab, Richmond, IL, USA). Mice in the sham surgery group received craniotomy but did not receive CCI, whereas mice in the naive control group received neither craniotomy nor hMSC-EVs treatment. For craniotomy, the mice were anesthetized with intramuscular injection of a cocktail comprising ketamine (30 mg/Kg), acepromazine (0.25 mg/Kg), and xylazine (3 mg/Kg). For inducing CCI, following anesthesia, the head of the mouse was fixed to a stereotactic frame (Kodali et al., 2023). Craniotomy (measuring ~4 mm in diameter) was performed on the right half of the calvaria after a midline incision. The region of craniotomy included an area overlying the temporoparietal cortex, with the center located midway between bregma and lambda and 2 mm away from the midline. A single impact was delivered to each mouse with a 3 mm diameter impactor tip, which penetrated 0.8 mm into the brain at a speed of 5 m/s and stayed for 300 milliseconds. Using tissue adhesive, a disk made from dental cement was placed on the craniotomy area, and surgical skin clips were used to close the skin. The animal was maintained in a temperature-controlled cage and monitored. The mice in the TBI group received IN administration of vehicle (PBS), whereas mice in the TBI + hMSC-EV groups received IN administration of 6.4, 12.8, or 25.6 × 10⁹ hMSC-EVs in PBS. To alleviate pain, all animals undergoing survival surgery received buprenorphine injections (0.1 mg/Kg, twice daily on days 1–2 and once daily on days 3–4). In addition, the mice were given 1 mL subcutaneous saline injections daily for 4 days to prevent dehydration.

The protocols employed for the culture and expansion of human bone marrow-derived hMSCs, and purification and characterization of hMSC-EVs, are described in the previous publications (Kim et al., 2016; Long et al., 2017; Kodali et al., 2023). Briefly, passage 4 (P4) hMSCs were cultured in a complete culture medium (CCM) and fed every 2 days. The CCM comprised alpha minimal essential medium (Invitrogen, Waltham, MA, USA), fetal bovine serum (Atlantic Biologicals, Miami, FL, USA), L-glutamine or glutamax (Invitrogen), and penicillin/streptomycin (Invitrogen). After ~70% confluence, the CCM was replaced with a commercially available serum-free medium (CD-CHO medium, Invitrogen) optimized with supplements. Six hours later, the medium was replaced with a fresh CD-CHO medium. After 48 h, the media from hMSC cultures were collected for the isolation of EVs. Harvested culture media were centrifuged to remove debris, filtered through 0.22 μm Stericups, aliquoted, and stored at −20^oC. After thawing, EVs in the media were isolated by anion-exchange chromatography (AEC), as detailed in the previously published reports (Kim et al., 2016; Kodali et al., 2023). NanoSight, a device used to measure the nano-sized particles (Malvern Panalytical, Malvern, UK), was used to measure the concentration and the size of EVs. The characterization of EVs for EV-specific marker expression using western blots and enzyme-linked immunoassays, ultrastructure using transmission electron microscopy, and antiinflammatory activity using lipopolysaccharide-stimulated mouse macrophage cultures are detailed in the previous studies (Kodali et al., 2023). The hMSC-EVs were intranasally dispensed bilaterally, as detailed in the previous publications (Kodali et al., 2019; Upadhya et al., 2020). Briefly, 1 h after TBI induction, each nostril was treated with hyaluronidase (100 U, Sigma-Aldrich, St. Louis, MO, USA) to enhance the permeability of the nasal mucous membrane. Thirty minutes later, the prep comprising hMSC-EVs was administered bilaterally via the IN route (5 μL/nostril each time).

The methods for brain tissue processing are similar to that described in the previous reports (Shetty et al., 2017; Kodali et al., 2018). In brief, animals were deeply anesthetized, perfused intracardially with 4% paraformaldehyde, and the brains removed. The brains were post-fixed overnight in 4% paraformaldehyde and processed for cryostat sectioning following cryoprotection with different concentrations of sucrose. The entire forebrain was sectioned coronally at thirty-micrometer thickness, and serial sections were collected and stored in an anti-freezing solution at −30 degrees centigrade. Every 15th section through the entire septo-temporal axis of the hippocampus was processed for BrdU or doublecortin (DCX) immunohistochemistry using avidin-biotin complex methods, as described in the previous reports (Hattiangady et al., 2008; Shetty et al., 2011). Five serial sections (every 15th) covered sampling from the entire hippocampus. The primary antibodies for visualizing BrdU+ newly generated cells and DCX+ immature neurons comprised a rat anti-BrdU (1:200 Abcam) and a goat anti-DCX (1:300, Abcam, Cambridge, MA, USA). The secondary antibodies comprised biotinylated anti-goat or anti-rat IgGs (1:250, Vector Labs, Burlingame, CA, USA). The study employed diaminobenzidine (Vector Labs) or vector SG (Vector Labs) as chromogens. After washing, the sections were mounted on 0.5% gelatin-coated slides, as described in the previous study.

Dual immunofluorescence procedures were employed to visualize BrdU+ cells expressing neuron-specific nuclear protein (NeuN) and pre-and post-synaptic proteins positive for synaptophysin (Syn) and post-synaptic density protein 95 (PSD95). These studies utilized three representative sections, one each from the anterior, mid, and posterior levels of the hippocampus. The detailed methods are available in the previous reports (Hattiangady and Shetty, 2010; Attaluri et al., 2022). The sections were washed in phosphate buffer saline (PBS), blocked with 10% normal donkey serum, and incubated overnight at 4°C in a cocktail of two primary antibodies (anti-rabbit NeuN and anti-rat BrdU or anti-rabbit Syn and anti-goat PSD95). The following day, the sections were treated with the appropriate secondary antibodies, rinsed in PBS, and coverslipped with a slow fade/antifade mounting medium (Invitrogen). The primary antibodies utilized were anti-rabbit NeuN (1:1000, Millipore Sigma, St. Louis, MO, USA), anti-rat BrdU (1:100, Abcam), anti-rabbit Syn (1:500, synaptic systems, Gottingen, Germany), and anti-goat PSD95 (1:500, Abcam). The secondary antibodies comprised donkey anti-rat IgG conjugated with Alexa Flour 594 (1:200, Invitrogen, Grand Island, NY, USA), donkey anti-rabbit IgG conjugated with Alexa Flour 488 (1:200, Invitrogen), donkey anti-goat IgG conjugated with Alexa Flour 594 (1:200, Invitrogen).

Every 15th thirty-micrometer section through the entire hippocampus was used for quantifying the numbers of BrdU+ newly born cells or DCX-positive newly born neurons in the SGZ-GCL of the hippocampus (n = 5 sections/marker/animal). A StereoInvestigator system (Microbrightfield, Williston, Vermont, USA) was employed to quantify BrdU+ and DCX+ cells using 100X objective lens and stereological methods, as detailed in the previous reports (Hattiangady et al., 2007; Kodali et al., 2021). The numbers of BrdU+ cells and DCX+ cells quantified using StereoInvestigator represent absolute counts for the entire SGZ-GCL of the hippocampus.

For quantifying the percentage of BrdU+ cells displaying NeuN expression in the SGZ-GCL, we employed Z-section analysis using a 40X objective lens in a Nikon confocal microscope. These studies utilized three representative sections, one each from the anterior, mid, and posterior levels of the hippocampus processed for BrdU-NeuN dual immunofluorescence (Shetty et al., 2012, 2020). In every animal, 25–30 BrdU+ cells were evaluated from 3 representative sections for NeuN expression (n = 5–6/group). Next, the numbers of new neurons born at post-TBI days 57–63 and survived for 3 weeks were determined for the hippocampus of TBI mice receiving vehicle or hMSC-EVs, age-matched naïve mice, and mice undergoing sham TBI via respective absolute counts of BrdU+ cells determined through stereology and percentages of BrdU+ cells expressing NeuN in the SGZ-GCL determined through Z-section analysis in a confocal microscope (Shetty et al., 2012, 2020). Thus, extrapolating the absolute numbers of BrdU+ cells with the respective percentages of BrdU+ cells expressing NeuN facilitated quantifying the total number of mature neurons added to the GCL over 7 days in various groups.

Z-sections obtained from three representative brain tissue sections stained for Syn and PSD95 dual immunofluorescence in a Nikon confocal microscope, and ImageJ, were employed to quantify area fractions of Syn and PSD95 in the somatosensory cortex (SSC) and the dentate molecular layer (Kodali et al., 2021). The images comprising Syn+ (green) or PSD95+ (red) puncta were first converted into an 8-bit grayscale image using Image J. Then, after thresholding, for each image comprising Syn+ or PSD95+ puncta, the area fractions of Syn+ or PSD95+ structures were measured. In addition, we also quantified putative synapses (i.e., the contact sites between Syn+ and PSD95+ structures) from randomly selected regions each measuring 2.15 μm². For each brain region, six Z-images collected from 3 representative sections from each animal (n = 5–6/group).

The frozen fresh brain samples were thawed, and the entire injured hippocampus (i.e., hippocampus ipsilateral to the CCI) from TBI mice receiving vehicle or hMSC-EVs was micro-dissected and placed in a tissue extraction reagent (Invitrogen, Waltham, MA) containing protease inhibitor (Sigma Aldrich, 1:100 dilution). The entire hippocampi from naïve controls were also micro-dissected and treated similarly. Each hippocampus was individually lysed using sonication for 15–20 s at 4°C. The resultant solution was centrifuged at 15,000 g for 10 min. The supernatant was then collected, aliquoted, and stored at −80°C for later use. Using kits, the concentrations of mature BDNF (R&D Systems, Minneapolis, MN, USA), p-ERK1/2, and p-CREB (Cell Signaling, Danvers, MA, USA) were measured from hippocampal lysates. In brief, hippocampal lysates were added to precoated 96-well plates with capture antibodies and incubated. After a thorough rinse in a wash buffer, specific biotin-conjugated detection antibodies were added and incubated at room temperature. Following the wash, the plates were incubated with streptavidin-HRP conjugate with gentle shaking. The plates were then treated with substrate solution, and the reaction was terminated by adding a stop solution before being measured at 450 nm. The concentration of each protein was determined using standard graphs. All protein values were normalized to 1 mg of total protein in the tissue lysate.

After determining the total protein concentration from SSC tissue lysates (n = 6/group) by BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA), 30 μg of protein were loaded onto 4–12% NuPAGE Bis-Tris Gels (ThermoFisher Scientific) and separated. After transferring proteins onto a nitrocellulose membrane using the iBlot2 gel transfer device (ThermoFisher Scientific), proteins in the membrane were detected using antibodies against Syn (1:5000, ProteinTech, Rosemont, IL, USA), PSD95 (1:1000, Abcam) and GAPDH (1:1000, Millipore). GAPDH was used as a loading control for cortex tissue lysates. Then, the protein signals were detected using the chemiluminescence reagents provided with the ECL detection kit (ThermoFisher Scientific) and visualized using the iBright imaging system (ThermoFisher Scientific). ImageJ software was used to determine the intensity of each targeted band (Syn, 38 kDa; PSD95: 95 kDa) by normalizing it to a corresponding GAPDH band, also run along with Syn and PSD95.

Power analysis using G*Power software suggested that for the effect size (delta) of 0.93 and an alpha of 0.05, 4 mice/group are sufficient to obtain a power of over 0.8. Therefore, we obtained final data from 4 to 6 mice in all assays in the study. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison post hoc tests was employed for detecting differences between three or more datasets. Furthermore, we conducted the non-parametric Kruskal-Wallis test, followed by Dunn’s post hoc tests when individual groups did not pass the normality test (Shapiro–Wilk test). p < 0.05 was taken as a statistically significant value in all measurements.

Through BrdU labeling and BrdU-NeuN dual immunofluorescence methods, we first evaluated the numbers of new neurons born in the injured hippocampus at post-TBI days 57–63 and survived for 3 weeks in TBI mice receiving vehicle or different doses of hMSC-EVs, in comparison to age-matched naïve control mice and sham TBI mice. Representative images showing the distribution of BrdU+ cells in the SGZ-GCL of different groups are illustrated in Figures 2A–C, G–I. Magnified images show the morphology of BrdU+ cells in different groups (Figures 2D–F, J–L). One-way ANOVA analysis of numbers of BrdU+ cells in the SGZ-GCL revealed no significant differences between naïve control mice, sham TBI mice, and TBI mice receiving vehicle or different doses of hMSC-EVs (p > 0.05, Figure 2M). Quantification of percentages of BrdU+ cells that expressed the mature neuronal marker NeuN in the SGZ-GCL enabled the quantification of net hippocampal neurogenesis. Representative images of BrdU+ cells differentiating into NeuN+ neurons in naïve, TBI, and TBI + EV groups (6.4–25.6 × 10⁹) are shown in Figures 3A–R. Notably, neuronal differentiation of newly generated cells diverged substantially between groups (p < 0.0001, Figure 3S). Compared to the naïve control group, the extent of neuronal differentiation was reduced in the TBI and TBI + 6.4 × 10⁹ EV group (p < 0.001–0.0001, Figure 3S). However, the neuronal differentiation of newly generated cells in TBI + 12.8 or 25.6 × 10⁹ EV groups was comparable to the naïve control group (p > 0.05, Figure 3S). Compared to the sham group also, the extent of neuronal differentiation was reduced in the TBI and TBI + 6.4 × 10⁹ EV group (p < 0.01–0.001, Figure 3S), but the degree of neuronal differentiation of newly generated cells in TBI + 12.8 or 25.6 × 10⁹ EV groups remained similar to the sham group (p > 0.05, Figure 3S).

Furthermore, the TBI mice that received higher doses of EVs (TBI + 12.8 or 25.6 × 10⁹) had significantly higher neuronal differentiation of newly generated cells than the TBI group (p < 0.05, Figure 3S). Because of the differences in neuronal differentiation, net hippocampal neurogenesis, computed by extrapolating the absolute number of BrdU+ cells for the entire SGZ-GCL (generated through stereological counts) with the percentage of BrdU+ cells that differentiated into NeuN+ neurons, also varied between groups (p < 0.001, Figure 3T). The net hippocampal neurogenesis was reduced in the TBI and TBI + 6.4 × 10⁹ EV groups compared to the naïve control group (p < 0.05–0.001, Figure 3T). However, the TBI + 12.8 or 25.6 × 10⁹ EV groups displayed comparable net neurogenesis as seen in the naïve control group (p > 0.05, Figure 3T). Compared to the sham group, net hippocampal neurogenesis was reduced in the TBI group (p < 0.01, Figure 3T), but net hippocampal neurogenesis in TBI + 6.4, 12.8 or 25.6 × 10⁹ EV groups remained similar to the sham group (p > 0.05, Figure 3T). Furthermore, the TBI + 25.6 × 10⁹ EV group displayed significantly better net neurogenesis than the TBI group (p < 0.05, Figure 3T). Thus, higher doses of hMSC-EV treatment in the acute phase of TBI preserved hippocampal neurogenesis at naïve control levels in the chronic phase of TBI.

We next quantified DCX+ newly born neurons in the SGZ-GCL to assess the status of hippocampal neurogenesis at 84 days post-TBI. Representative images showing the distribution of DCX+ neurons in different groups are presented (Figures 4A–C, G–I). Magnified images revealed similar morphology of DCX+ neurons across groups (Figures 4D–F, J–L). Notably, signs of abnormal neurogenesis (i.e., the migration of newly born neurons into the dentate hilus or the dentate molecular layer were not seen in any of the groups). The dendrites of most neurons in all groups projected through the GCL into the inner and middle molecular layers of the dentate gyrus (Figures 4A–L). ANOVA analysis of the numbers of DCX+ neurons in the SGZ-GCL revealed significant differences between groups (p < 0.0001, Figure 4M). The DCX+ newly born neurons were reduced in the TBI and TBI + 6.4 × 10⁹ EV group compared to the naïve control group (p < 0.001, Figure 4M). In contrast, DCX+ neurons in the TBI + 12.8 or 25.6 × 10⁹ EV groups matched numbers in the naïve control group (p > 0.05, Figure 4M). The TBI + 12.8 or 25.6 × 10⁹ EV groups also displayed significantly higher DCX+ newly born neuron numbers than the TBI group (p < 0.05–0.0001, Figure 4M). Thus, higher doses of hMSC-EV treatment after TBI preserved the status of hippocampal neurogenesis at naïve control levels at 84 days post-TBI.

Next, we investigated whether a single IN treatment of 25.6 × 10⁹ hMSC-EVs 90 min after TBI would ease the downregulation of the BDNF-ERK-CREB signaling pathway in the hippocampus ipsilateral to injury in the acute phase of TBI. We quantified the concentrations of major components of the BDNF-ERK-CREB signaling pathway, such as BDNF, pERK1/2, and p-CREB in the hippocampus, to grasp the potential mechanisms of hMSC-EV treatment-mediated maintenance of hippocampal neurogenesis at naïve control levels. ANOVA analysis uncovered differences between groups for pERK1/2, p-CREB, and BDNF levels (p < 0.05–0.01, Figures 5A–C). The TBI group exhibited a diminished concentration of pERK1/2 vis-à-vis the naïve control group (p < 0.05, Figure 5A). hMSC-EV treatment after TBI normalized pERK1/2 concentration to naïve control levels (p > 0.05, Figure 5A), but the EV treatment-mediated increase did not go significantly above the TBI group (p > 0.05, Figure 5A). A reduced p-CREB concentration was observed in the TBI group vis-à-vis the naïve control group (p < 0.05, Figure 5B). hMSC-EV treatment boosted the concentration of p-CREB. The overall increase did not statistically differ from the concentration in the naïve control group (p > 0.05, Figure 5B) but was enhanced compared to the TBI group (p < 0.01, Figure 5B). BDNF concentration was reduced in the TBI group compared to the naïve control group (p < 0.01, Figure 5C). hMSC-EV treatment increased the BDNF concentration closer to naïve control levels (p > 0.05, Figure 5C). However, the EV treatment-mediated augmentation did not rise significantly above the TBI group (p > 0.05, Figure 5C).

We also measured BDNF concentration in the hippocampus at 84 days post-EV treatment to address whether the increased concentration observed at 48 h post-EV treatment was sustained at 84 days post-EV treatment. This analysis showed that the hippocampus of TBI mice receiving EVs displayed BDNF concentration equivalent to the age-matched naïve control mice (p > 0.05, Figure 5D). In contrast, TBI mice receiving the vehicle displayed reduced BDNF concentration compared to naïve control mice (p < 0.05, Figure 5D). However, BDNF levels did not differ significantly between TBI and TBI + EVs groups (p > 0.05, Figure 5D). Overall, hMSC-EV treatment after TBI brought the concentrations of p-ERK1/2, p-CREB, and BDNF closer to naïve control levels in the acute phase of TBI. Furthermore, increased BDNF concentrations in TBI mice receiving EVs observed in the acute phase of TBI were sustained in the chronic phase of TBI. However, p-ERK1/2 and BDNF levels in the acute phase and BDNF concentration in the chronic phase did not differ significantly between TBI and TBI + EVs groups, suggesting a partial recovery of the BDNF-ERK-CREB signaling with hMSC-EV treatment.

We evaluated Syn+ and PSD95+ puncta in the molecular layer of the dentate gyrus and SSC, ipsilateral to CCI. We included SSC in Syn+ and PSD95+ puncta measurements to ascertain potential changes in the synaptic density in the peri-injury area. Representative images of Syn+ and PSD95+ puncta from the dentate molecular layer and SSC, along with potential synapses, are illustrated (Figures 6A–T, 7A–T). ANOVA analysis of area fractions of Syn+ and PSD95+ puncta and putative synapses in both regions revealed significant differences between groups (p < 0.05–0.01, Figures 6U–W, 7U–W). The area fractions of Syn+ puncta were reduced in the TBI group compared to the naïve control group in both regions (p < 0.05, Figures 6U, 7U). In contrast, TBI + 6.4, 12.8 or 25.6 × 10⁹ EV groups, Syn+ puncta were equivalent to the naïve control group (p > 0.05, Figures 6U, 7U). The TBI + 25.6 × 10⁹ EV group also displayed significantly higher Syn+ puncta than the TBI group in both regions (p < 0.05, Figures 6U, 7U).

Consistent with the above results, ANOVA analysis of area fractions of PSD95+ puncta and putative synapses in both the molecular layer of the dentate gyrus and SSC revealed significant differences between groups (p < 0.05–0.01, Figures 6V,W, 7V,W). In the dentate molecular layer, post hoc tests revealed no differences in the density of PSD95+ puncta or putative synapses between the naïve group and different TBI groups (p > 0.05, Figures 6V,W). However, the TBI + 25.6 × 10⁹ EV group displayed a higher density of PSD95+ puncta and putative synapses than the TBI group (p < 0.05–0.001, Figures 6V,W).

In contrast, in SSC, the area fractions of PSD95+ puncta and putative synapses were reduced in the TBI group compared to the naïve control group (p < 0.05, Figures 7V,W). However, in TBI + 6.4, 12.8, or 25.6 × 10⁹ EV groups, PSD95+ puncta were equivalent to the naïve control group (p > 0.05, Figure 7V). The TBI + 25.6 × 10⁹ EV group also displayed significantly higher PSD95+ puncta than the TBI group (p < 0.05, Figure 6V).

Moreover, in TBI + 12.8 or 25.6 × 10⁹ EV groups, putative synapses were equivalent to the naïve control group (p > 0.05, Figure 7W). Thus, hMSC-EV treatment after TBI eased the loss of synapses in both the dentate molecular layer and SSC.

Furthermore, western blot analysis of SSC lysates confirmed a significant reduction in both Syn and PSD95 protein levels in TBI mice receiving vehicle compared to naive control mice (p < 0.05–0.01, Figures 8A–C). However, TBI mice receiving hMSC-EVs displayed Syn and PSD95 levels closer to naive control mice (p > 0.05, Figures 8A–C). Thus, western blot results validate the findings of Syn+ and PSD95+ puncta measurements from the SSC.