Cortical injury, such as injuries after stroke or age-related ischemic events, triggers a cascade of degeneration accompanied by inflammatory responses that mediate neurological deficits. Therapeutics that modulate such neuroinflammatory responses in the aging brain have the potential to reduce neurological dysfunction and promote recovery. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) are lipidbound, nanoscale vesicles that can modulate inflammation and enhance recovery in rodent stroke models. We recently assessed the efficacy of intravenous infusions of MSC-EVs (24-h and 14-days post-injury) as a treatment in aged rhesus monkeys (Macaca mulatta) with cortical injury that induced impairment of fine motor function of the hand. Aged monkeys treated with EVs after injury recovered motor function more rapidly and more fully than aged monkeys given a vehicle control. Here, we describe EV-mediated inflammatory changes using histological assays to quantify differences in markers of neuroinflammation in brain tissue between EV and vehicle-treated aged monkeys. The activation status of microglia, the innate macrophages of the brain, is critical to cell fate after injury. Our findings demonstrate that EV treatment after injury is associated with greater densities of ramified, homeostatic microglia, along with reduced pro-inflammatory microglial markers. These findings are consistent with a phenotypic switch of inflammatory hypertrophic microglia towards anti-inflammatory, homeostatic functions, which was correlated with enhanced functional recovery. Overall, our data suggest that EVs reduce neuroinflammation and shift microglia towards restorative functions. These findings demonstrate the therapeutic potential of MSC-derived EVs for reducing neuroinflammation after cortical injury in the aged brain.

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Functional recovery after cortical injury, such as stroke, is associated with neural circuit reorganization, but the underlying mechanisms and efficacy of therapeutic interventions promoting neural plasticity in primates are not well understood. Bone marrow mesenchymal stem cell-derived extracellular vesicles (MSC-EVs), which mediate cell-to-cell inflammatory and trophic signaling, are thought be viable therapeutic targets. We recently showed, in aged female rhesus monkeys, that systemic administration of MSC-EVs enhances recovery of function after injury of the primary motor cortex, likely through enhancing plasticity in perilesional motor and premotor cortices. Here, using in vitro whole-cell patch-clamp recording and intracellular filling in acute slices of ventral premotor cortex (vPMC) from rhesus monkeys (Macaca mulatta) of either sex, we demonstrate that MSC-EVs reduce injury-related physiological and morphologic changes in perilesional layer 3 pyramidal neurons. At 14-16 weeks after injury, vPMC neurons from both vehicle-and EV-treated lesioned monkeys exhibited significant hyperexcitability and predominance of inhibitory synaptic currents, compared with neurons from nonlesioned control brains. However, compared with vehicle-treated monkeys, neurons from EV-treated monkeys showed lower firing rates, greater spike frequency adaptation, and excitatory:inhibitory ratio. Further, EV treatment was associated with greater apical dendritic branching complexity, spine density, and inhibition, indicative of enhanced dendritic plasticity and filtering of signals integrated at the soma. Importantly, the degree of EV-mediated reduction of injury-related pathology in vPMC was significantly correlated with measures of behavioral recovery. These data show that EV treatment dampens injury-related hyperexcitability and restores excitatory:inhibitory balance in vPMC, thereby normalizing activity within cortical networks for motor function.

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Significance: Myelin breakdown is likely a key factor in the loss of cognitive and motor function associated with many neurodegenerative diseases. Aim: New methods for imaging myelin structure are needed to characterize and quantify the degradation of myelin in standard whole-brain sections of nonhuman primates and in human brain. Approach: Quantitative birefringence microscopy (qBRM) is a label-free technique for rapid histopathological assessment of myelin structural breakdown following cortical injury in rhesus monkeys. Results: We validate birefringence microscopy for structural imaging of myelin in rhesus monkey brain sections, and we demonstrate the power of qBRM by characterizing the breakdown of myelin following cortical injury, as a model of stroke, in the motor cortex. Conclusions: Birefringence microscopy is a valuable tool for histopathology of myelin and for quantitative assessment of myelin structure. Compared to conventional methods, this label-free technique is sensitive to subtle changes in myelin structure, is fast, and enables more quantitative assessment, without the variability inherent in labeling procedures such as immunohistochemistry.

Exosomes are extracellular vesicles that mediate cell‐to‐cell communication and have promising therapeutic effects for cortical injury caused by stroke or trauma. Accordingly, we tested exosomes derived from mesenchymal stem cells as a therapy to enhance recovery in our nonhuman primate model of cortical injury. Aged female monkeys were trained on fine motor hand tasks and randomly assigned to exosome (n=5) or a vehicle control (PBS) (n=5) groups. A lesion was then made in the hand representation of the primary motor cortex (M1). At 24 hours and 14 days post‐injury, monkeys were each given 4×1011 exosomes/kg or vehicle. At 15 days post‐injury, post‐operative testing on motor tasks resumed for 12 weeks. Treated monkeys all demonstrated a greater degree of functional recovery than vehicle controls. To assess mechanisms underlying exosome‐mediated enhancement of recovery, serum, cerebrospinal fluid (CSF), and terminal brain tissue were analyzed for markers of neuronal damage and neuroinflammation. To assess neuronal damage, we quantified Myelin Basic Protein (MBP) in CSF using ELISA, which showed a sharp increase 24‐hours after injury across all monkeys, followed by a decline in both groups. However, the decline in [MBP] was more rapid in exosome treated monkeys compared to vehicle, suggesting that exosomes had an immediate effect to reduce this marker of cortical injury. In addition, ELISA analysis of serum showed decreased RANTES (p=0.04) and Eotaxin (p=0.03) in exosome‐treated monkeys, suggesting an exosome‐mediated reduction of inflammation across the recovery period. To assess whether exosomes affected cellular markers of inflammation in the brain, we characterized microglial protein expression and morphology using immunohistochemistry and confocal microscopy. Optical density quantification of microglial markers Iba1, P2RY12, and the MHCII antigen‐presenting marker, LN3, showed that exosome‐treated animals had significantly lower expression (p=0.019) and smaller LN3+ puncta sizes (p=0.028). Sholl analysis of microglial morphology showed greater ramification length (p=0.019), number of intersections (p=0.02), and 3D convex‐hull volume of coverage (p=0.016) in exosome treated monkeys. Quantification of microglial cell density by morphology and LN3 expression further showed that exosome‐treatment reduced the number of hypertrophic “inflammatory” LN3+ microglia (p<0.05) and increased ramified microglia (LN3− and LN3+) (p<0.05). These results demonstrate that exosome treatment significantly reduces injury related damage and biomarkers of peripheral and brain inflammation, providing compelling data for the translational value of exosomes as a treatment for human stroke patients. Support or Funding Information Supported by NIH grant R21NS102991‐02 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cortical injury, such as injury from stroke, results in a cascade of events that includes cell death, inflammation and disruption of myelin. To date, there are no highly effective treatments for reducing the deficits that occur after injury. Recently, we have demonstrated that extracellular vesicles (EVs) harvested from rhesus monkey bone marrow derived cells when given 1 day and 14 days following injury facilitate recovery of function in aged rhesus monkeys within the first 3–5 weeks after cortical injury. Based on these findings and current proteomic literature of MSC‐EVs, we hypothesized that MSC‐EVs enhance myelin plasticity by limiting damage to oligodendrocytes and stimulating remyelination. To assess general myelin integrity after injury, we used Spectral Confocal Reflectance Microscopy (SCoRe) to image myelinated axons and found an increase in the density of myelinated axons in the EV group (p < 0.05). To assess whether the difference was due to reduced damage or remyelination, in sublesional white matter we assessed immunohistochemical labeling of Olig2, a general oligodendrocyte marker, and 8OHdG, a marker for DNA damage. We found reduced densities of Olig2 colocalized with 8OHdG in the EV group (p<0.05). As a marker of active demyelination and myelin debris clearance, we measured Myelin Basic Protein (MBP) concentrations in CSF and found a longitudinal reduction in the EV animals. To assess remyelination, we measured expression of MBP, a gene for myelination in mature oligodendrocytes, Myelin Regulatory Factor (MyRF), a gene for oligodendrocyte differentiation and maintenance, and Breast Carcinoma Amplified Sequence 1 (BCAS1), a gene for newly myelinating oligodendrocytes. Interestingly, we found a 4 fold increase in MyRF expression, and a 1.5 fold increase in MBP and BCAS1 in the EV animals relative to the vehicle control animals in perilesional brain tissue. Consistent with these gene expression differences associated with re‐myelination, we found that the densities of newly‐myelinating oligodendrocytes immune‐labeled with BCAS1, as well as mature oligodendrocytes expressing CC1, exhibited a trend towards an increase in the EV group (p = 0.09). These results suggest that EV treatment reduces myelin damage, while also stimulating myelin repair. Finally, these results correlated with enhanced motor recovery, suggesting that EV‐mediated white matter plasticity is a critical component for recovery after cortical injury. Support or Funding Information Supported by NIH grant R21NS102991‐02.

Exosomes are extracellular vesicles that mediate cell‐to‐cell communication and have promising therapeutic effects for cortical injury caused by stroke or trauma. Accordingly, we tested exosomes derived from mesenchymal stem cells as a therapy to enhance recovery in our nonhuman primate model of cortical injury. Aged female monkeys were trained on fine motor hand tasks and randomly assigned to exosome (n=5) or a vehicle control (PBS) (n=5) groups. A lesion was then made in the hand representation of the primary motor cortex (M1). At 24 hours and 14 days post‐injury, monkeys were each given 4×1011 exosomes/kg or vehicle. At 15 days post‐injury, post‐operative testing on motor tasks resumed for 12 weeks. Treated monkeys all demonstrated a greater degree of functional recovery than vehicle controls. To assess mechanisms underlying exosome‐mediated enhancement of recovery, serum, cerebrospinal fluid (CSF), and terminal brain tissue were analyzed for markers of neuronal damage and neuroinflammation. To assess neuronal damage, we quantified Myelin Basic Protein (MBP) in CSF using ELISA, which showed a sharp increase 24‐hours after injury across all monkeys, followed by a decline in both groups. However, the decline in [MBP] was more rapid in exosome treated monkeys compared to vehicle, suggesting that exosomes had an immediate effect to reduce this marker of cortical injury. In addition, ELISA analysis of serum showed decreased RANTES (p=0.04) and Eotaxin (p=0.03) in exosome‐treated monkeys, suggesting an exosome‐mediated reduction of inflammation across the recovery period. To assess whether exosomes affected cellular markers of inflammation in the brain, we characterized microglial protein expression and morphology using immunohistochemistry and confocal microscopy. Optical density quantification of microglial markers Iba1, P2RY12, and the MHCII antigen‐presenting marker, LN3, showed that exosome‐treated animals had significantly lower expression (p=0.019) and smaller LN3+ puncta sizes (p=0.028). Sholl analysis of microglial morphology showed greater ramification length (p=0.019), number of intersections (p=0.02), and 3D convex‐hull volume of coverage (p=0.016) in exosome treated monkeys. Quantification of microglial cell density by morphology and LN3 expression further showed that exosome‐treatment reduced the number of hypertrophic “inflammatory” LN3+ microglia (p<0.05) and increased ramified microglia (LN3− and LN3+) (p<0.05). These results demonstrate that exosome treatment significantly reduces injury related damage and biomarkers of peripheral and brain inflammation, providing compelling data for the translational value of exosomes as a treatment for human stroke patients.Support or Funding InformationSupported by NIH grant R21NS102991‐02This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.