Quantitative birefringence microscopy for imaging the structural integrity of CNS myelin following circumscribed cortical injury in the rhesus monkey.

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.

Extracellular vesicles derived from bone marrow mesenchymal stem cells enhance myelin maintenance after cortical injury in aged rhesus monkeys.

Cortical injury, such as stroke, causes neurotoxic cascades that lead to rapid death and/or damage to neurons and glia. Axonal and myelin damage in particular, are critical factors that lead to neuronal dysfunction and impair recovery of function after injury. These factors can be exacerbated in the aged brain where white matter damage is prevalent. Therapies that can ameliorate myelin damage and promote repair by targeting oligodendroglia, the cells that produce and maintain myelin, may facilitate recovery after injury, especially in the aged brain where these processes are already compromised. We previously reported that a novel therapeutic, Mesenchymal Stem Cell derived extracellular vesicles (MSC-EVs), administered intravenously at both 24 h and 14 days after cortical injury, reduced microgliosis (Go et al. 2019), reduced neuronal pathology (Medalla et al. 2020), and improved motor recovery (Moore et al. 2019) in aged female rhesus monkeys. Here, we evaluated the effect of MSC-EV treatment on changes in oligodendrocyte maturation and associated myelin markers in the sublesional white matter using immunohistochemistry, confocal microscopy, stereology, qRT-PCR, and ELISA. Compared to vehicle control monkeys, EV-treated monkeys showed a reduction in the density of damaged oligodendrocytes. Further, EV-treatment was associated with enhanced myelin maintenance, evidenced by upregulation of myelin-related genes and increases in actively myelinating oligodendrocytes in sublesional white matter. These changes in myelination correlate with the rate of motor recovery, suggesting that improved myelin maintenance facilitates this recovery. Overall, our results suggest that EVs act on oligodendrocytes to support myelination and improves functional recovery after injury in the aged brain. SIGNIFICANCE: We previously reported that EVs facilitate recovery of function after cortical injury in the aged monkey brain, while also reducing neuronal pathology (Medalla et al. 2020) and microgliosis (Go et al. 2019). However, the effect of injury and EVs on oligodendrocytes and myelination has not been characterized in the primate brain (Dewar et al. 1999; Sozmen et al. 2012; Zhang et al. 2013). In the present study, we assessed changes in myelination after cortical injury in aged monkeys. Our results show, for the first time, that MSC-EVs support recovery of function after cortical injury by enhancing myelin maintenance in the aged primate brain.

Treatment with Mesenchymal-Derived Extracellular Vesicles Reduces Injury-Related Pathology in Pyramidal Neurons of Monkey Perilesional Ventral Premotor Cortex.

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.SIGNIFICANCE STATEMENT Neuronal plasticity can facilitate recovery of function after cortical injury, but the underlying mechanisms and efficacy of therapeutic interventions promoting this plasticity in primates are not well understood. Our recent work has shown that intravenous infusions of mesenchymal-derived extracellular vesicles (EVs) that are involved in cell-to-cell inflammatory and trophic signaling can enhance recovery of motor function after injury in monkey primary motor cortex. This study shows that this EV-mediated enhancement of recovery is associated with amelioration of injury-related hyperexcitability and restoration of excitatory-inhibitory balance in perilesional ventral premotor cortex. These findings demonstrate the efficacy of mesenchymal EVs as a therapeutic to reduce injury-related pathologic changes in the physiology and structure of premotor pyramidal neurons and support recovery of function.

Extracellular vesicles from mesenchymal stem cells reduce microglial-mediated neuroinflammation after cortical injury in aged Rhesus monkeys.

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 lipid-bound, 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.