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.

Alterations in high-order diffusion imaging in veterans with Gulf War Illness is associated with chemical weapons exposure and mild traumatic brain injury.

The complex etiology behind Gulf War Illness (GWI) has been attributed to the combined exposure to neurotoxicant chemicals, brain injuries, and some combat experiences. Chronic GWI symptoms have been shown to be associated with intensified neuroinflammatory responses in animal and human studies. To investigate the neuroinflammatory responses and potential causes in Gulf War (GW) veterans, we focused on the effects of chemical/biological weapons (CBW) exposure and mild traumatic brain injury (mTBI) during the war. We applied a novel MRI diffusion processing method, Neurite density imaging (NDI), on high-order diffusion imaging to estimate microstructural alterations of brain imaging in Gulf War veterans with and without GWI, and collected plasma proinflammatory cytokine samples as well as self-reported health symptom scores. Our study identified microstructural changes specific to GWI in the frontal and limbic regions due to CBW and mTBI, and further showed distinctive microstructural patterns such that widespread changes were associated with CBW and more focal changes on diffusion imaging were observed in GW veterans with an mTBI during the war. In addition, microstructural alterations on brain imaging correlated with upregulated blood proinflammatory cytokine markers TNFRI and TNFRII and with worse outcomes on self-reported symptom measures for fatigue and sleep functioning. Taken together, these results suggest TNF signaling mediated inflammation affects frontal and limbic regions of the brain, which may contribute to the fatigue and sleep symptoms of the disease and suggest a strong neuroinflammatory component to GWI. These results also suggest exposures to chemical weapons and mTBI during the war are associated with different patterns of peripheral and central inflammation and highlight the brain regions vulnerable to further subtle microscale morphological changes and chronic signaling to nearby glia.

Corticosterone potentiates DFP-induced neuroinflammation and affects high-order diffusion imaging in a rat model of Gulf War Illness.

Veterans of the 1991 Gulf War were potentially exposed to a variety of toxic chemicals, including sarin nerve agent and pesticides, which have been suspected to be involved in the development of Gulf War Illness (GWI). Several of these exposures cause a neuroinflammatory response in mice, which may serve as a basis for the sickness behavior-like symptoms seen in veterans with GWI. Furthermore, conditions mimicking the physiological stress experienced during the war can exacerbate this effect. While neuroinflammation has been observed post-exposure using animal models, it remains a challenge to evaluate neuroinflammation and its associated cellular and molecular changes in vivo in veterans with GWI. Here, we evaluated neuroimmune-associated alterations in intact brains, applying our existing GWI mouse model to rats, by exposing them to 4days of corticosterone (CORT; 200mg/L in the drinking water), to mimic high physiological stress, followed by a single injection of the sarin nerve agent surrogate, diisopropyl fluorophosphate (DFP; 1.5mg/kg, i.p.). Then, we evaluated the neuroinflammatory responses using qPCR of cytokine mRNA and also examined brain structure with a novel high-order diffusion MRI. We found a CORT-enhancement of DFP-induced neuroinflammation, extending our mouse GWI model to the rat. High order diffusion MRI revealed different patterns among the different treatment groups. Particularly, while the CORT+DFP rats had more restricted spatial patterns in the hippocampus and the hypothalamus, the highest and most wide-spread differences were shown in DFP-treated rats compared to the controls in the thalamus, the amygdala, the piriform cortex and the ventral tegmental area. The association of these diffusion changes with neuroinflammatory cytokine expression indicates the potential for GW-relevant exposures to result in connectivity changes in the brain. By transferring this high order diffusion MRI into in vivo imaging in veterans with GWI, we can achieve further insights on the trajectories of the neuroimmune response over time and its impacts on behavior and potential neurological damage.

Oral curcumin supplementation improves fine motor function in the middle-aged rhesus monkey.

Aged individuals experience decreased fine motor function of the hand and digits, which could result, in part, from the chronic, systemic state of inflammation that occurs with aging. Recent research for treating age-related inflammation has focused on the effects of nutraceuticals that have anti-inflammatory properties. One particular dietary polyphenol, curcumin, the principal curcuminoid of the spice turmeric, has been shown to have significant anti-inflammatory effects and there is mounting evidence that curcumin may serve to reduce systemic inflammation. Therefore, it could be useful for alleviating age-related impairments in fine motor function. To test this hypothesis we assessed the efficacy of a dietary intervention with a commercially available optimized curcumin to ameliorate or delay the effects of aging on fine motor function of the hand of rhesus monkeys. We administered oral daily doses of curcumin or a control vehicle to 11 monkeys over a 14- to 18-month period in which they completed two rounds of fine motor function testing. The monkeys receiving curcumin were significantly faster at retrieving a food reward by round 2 of testing than monkeys receiving a control vehicle. Further, the monkeys receiving curcumin demonstrated a greater degree of improvement in performance on our fine motor task by round 2 of testing than monkeys receiving a control vehicle. These findings reveal that fine motor function of the hand and digits is improved in middle-aged monkeys receiving chronic daily administration of curcumin.

Substantia Nigra Pathology, Contact Sports Play, and Parkinsonism in Chronic Traumatic Encephalopathy.

Importance: Parkinsonism is associated with traumatic brain injury and chronic traumatic encephalopathy (CTE), a neurodegenerative disease associated with repetitive head impact (RHI) exposure, but the neuropathologic substrates that underlie parkinsonism in individuals with CTE are yet to be defined. Objective: To evaluate the frequency of parkinsonism in individuals with CTE and the association of RHI and neuropathologic substrates with parkinsonism in these individuals. Design, Setting, and Participants: This cross-sectional study included brain donors with neuropathologically diagnosed CTE without other significant neurodegenerative disease and with information on parkinsonism from the Understanding Neurologic Injury and Traumatic Encephalopathy brain bank between July 2015 and May 2022. Exposure: Years of contact sports participation as a proxy for RHI. Main Outcomes and Measures: The main outcomes were frequency of parkinsonism in individuals with CTE and associations between (1) RHI with substantia nigra (SN) Lewy bodies (LBs) and neurofibrillary tangles (NFTs); (2) LBs, NFTs, and arteriolosclerosis with SN neuronal loss; and (3) SN neuronal loss, LBs, NFTs, and arteriolosclerosis with parkinsonism, tested by age-adjusted logistic regressions. Results: Of 481 male brain donors with neuropathologically diagnosed CTE, parkinsonism occurred frequently in individuals with CTE (119 [24.7%]; 362 [75.3%] did not have parkinsonism). Participants with parkinsonism had a higher mean (SD) age at death (71.5 [13.0] years) than participants without parkinsonism (54.1 [19.3] years) (P < .001) and higher rates of dementia (104 [87.4%] vs 105 [29.0%]), visual hallucinations (45 [37.8%] vs 51 [14.1%]), and probable rapid eye movement sleep behavior disorder (52 [43.7%] vs 58 [16.0%]) (P < .001 for all). Participants with parkinsonism had a more severe CTE stage (eg, stage IV: 35 [29.4%] vs 39 [10.8%]) and nigral pathology than those without parkinsonism (NFTs: 50 of 117 [42.7%] vs 103 of 344 [29.9%]; P = .01; neuronal loss: 61 of 117 [52.1%] vs 59 of 344 [17.1%]; P < .001; and LBs: 28 of 116 [24.1%] vs 20 of 342 [5.8%]; P < .001). Years of contact sports participation were associated with SN NFTs (adjusted odds ratio [AOR], 1.04; 95% CI, 1.00-1.07; P = .03) and neuronal loss (AOR, 1.05; 95% CI, 1.01-1.08; P = .02). Nigral neuronal loss (AOR, 2.61; 95% CI, 1.52-4.47; P < .001) and LBs (AOR, 2.29; 95% CI, 1.15-4.57; P = .02) were associated with parkinsonism. However, SN neuronal loss was associated with SN LBs (AOR, 4.48; 95% CI, 2.25-8.92; P < .001), SN NFTs (AOR, 2.51; 95% CI, 1.52-4.15; P < .001), and arteriolosclerosis (AOR, 2.27; 95% CI, 1.33-3.85; P = .002). In American football players, regression analysis demonstrated that SN NFTs and neuronal loss mediated the association between years of play and parkinsonism in the context of CTE (ß, 0.012; 95% CI, 0.001-0.038). Conclusions and Relevance: In this cross-sectional study of contact sports athletes with CTE, years of contact sports participation were associated with SN tau pathology and neuronal loss, and these pathologies were associated with parkinsonism. Repetitive head impacts may incite neuropathologic processes that lead to symptoms of parkinsonism in individuals with CTE.

Neural recovery after cortical injury: Effects of MSC derived extracellular vesicles on motor circuit remodeling in rhesus monkeys.

Reorganization of motor circuits in the cortex and corticospinal tract are thought to underlie functional recovery after cortical injury, but the mechanisms of neural plasticity that could be therapeutic targets remain unclear. Recent work from our group have shown that systemic treatment with mesenchymal stem cell derived (MSCd) extracellular vesicles (EVs) administered after cortical damage to the primary motor cortex (M1) of rhesus monkeys resulted in a robust recovery of fine motor function and reduced chronic inflammation. Here, we used immunohistochemistry for cfos, an activity-dependent intermediate early gene, to label task-related neurons in the surviving primary motor and premotor cortices, and markers of axonal and synaptic plasticity in the spinal cord. Compared to vehicle, EV treatment was associated with a greater density of cfos+ pyramidal neurons in the deep layers of M1, greater density of cfos+ inhibitory interneurons in premotor areas, and lower density of synapses on MAP2+ lower motor neurons in the cervical spinal cord. These data suggest that the anti-inflammatory effects of EVs may reduce injury-related upper motor neuron damage and hyperexcitability, as well as aberrant compensatory re-organization in the cervical spinal cord to improve motor function.

Distribution and overlap of entorhinal, premotor, and amygdalar connections in the monkey anterior cingulate cortex.

The anterior cingulate cortex (ACC) is important for decision-making as it integrates motor plans with affective and contextual limbic information. Disruptions in these networks have been observed in depression, bipolar disorder, and post-traumatic stress disorder. Yet, overlap of limbic and motor connections within subdivisions of the ACC is not well understood. Hence, we administered a combination of retrograde and anterograde tracers into structures important for contextual memories (entorhinal cortex), affective processing (amygdala), and motor planning (dorsal premotor cortex) to assess overlap of labeled projection neurons from (outputs) and axon terminals to (inputs) the ACC of adult rhesus monkeys (Macaca mulatta). Our data show that entorhinal and dorsal premotor cortical (dPMC) connections are segregated across ventral (A25, A24a) and dorsal (A24b,c) subregions of the ACC, while amygdalar connections are more evenly distributed across subregions. Among all areas, the rostral ACC (A32) had the lowest relative density of connections with all three regions. In the ventral ACC, entorhinal and amygdalar connections strongly overlap across all layers, especially in A25. In the dorsal ACC, outputs to dPMC and the amygdala strongly overlap in deep layers. However, dPMC input to the dorsal ACC was densest in deep layers, while amygdalar inputs predominantly localized in upper layers. These connection patterns are consistent with diverse roles of the dorsal ACC in motor evaluation and the ventral ACC in affective and contextual memory. Further, distinct laminar circuits suggest unique interactions within specific ACC compartments that are likely important for the temporal integration of motor and limbic information during flexible goal-directed behavior.

Cell based therapy reduces secondary damage and increases extent of microglial activation following cortical injury.

Cortical injury elicits long-term cytotoxic and cytoprotective mechanisms within the brain and the balance of these pathways can determine the functional outcome for the individual. Cytotoxicity is exacerbated by production of reactive oxygen species, accumulation of iron, and peroxidation of cell membranes and myelin. There are currently no neurorestorative treatments to aid in balancing the cytotoxic and cytoprotective mechanisms following cortical injury. Cell based therapies are an emerging treatment that may function in immunomodulation, reduction of secondary damage, and reorganization of surviving structures. We previously evaluated human umbilical tissue-derived cells (hUTC) in our non-human primate model of cortical injury restricted to the hand area of primary motor cortex. Systemic hUTC treatment resulted in significantly greater recovery of fine motor function compared to vehicle controls. Here we investigate the hypothesis that hUTC treatment reduces oxidative damage and iron accumulation and increases the extent of the microglial response to cortical injury. To test this, brain sections from these monkeys were processed using immunohistochemistry to quantify oxidative damage (4-HNE) and activated microglia (LN3), and Prussian Blue to quantify iron. hUTC treated subjects exhibited significantly reduced oxidative damage in the sublesional white matter and iron accumulation in the perilesional area as well as a significant increase in the extent of activated microglia along white matter pathways. Increased perilesional iron accumulation was associated with greater perilesional oxidative damage and larger reconstructed lesion volume. These findings support the hypothesis that systemic hUTC administered 24 h after cortical damage decreases the cytotoxic response while increasing the extent of microglial activation.

Long-term effects of curcumin in the non-human primate brain.

Curcumin has recently been shown to be a potential treatment for slowing or ameloriating cognitive decline during aging in our nonhuman primate model of normal aging. In these same monkeys, we studied for the first time the neurological impacts of long-term curcumin treatments using longitudinal magnetic resonance imaging (MRI). Sixteen rhesus monkeys received curcumin or a vehicle control for 14-18 months. We applied a combination of structural and diffusion MRI to determine whether the curcumin resulted in structural or functional changes in focal regions of the brain. The longitudinal imaging revealed decreased microscale diffusivity (mD) measurements mainly in the hippocampus and basal forebrain structures of curcumin treated animals. Changes in generalized fractional anisotropy (GFA) and grey matter density (GMd) measurements indicated an increased grey matter density in cortical ROIs with improved white matter integrity in limbic, cerebellar, and brain stem regions. These findings suggest that noticeable changes in the neuronal environment could be induced from long-term curcumin treatments. Results may provide a neurological basis on the recent findings demonstrating improved spatial working memory and motor function in nonhuman primates.

Microglia activation and phagocytosis: relationship with aging and cognitive impairment in the rhesus monkey.

While cognitive decline is observed in the normal aging monkey, neurons are not lost with age. Instead, frontal white matter is lost as myelin degenerates and both correlate with age-related cognitive decline. As age-related myelin damage increases, there should be an increase in clearance of damaged myelin by microglial phagocytosis. In this study, brains of behaviorally tested rhesus monkeys were assessed using unbiased stereology to quantify the density of activated microglia (LN3 antibody positive) and phagocytic microglia (galectin-3 (Gal-3) antibody positive) in three white matter regions: the corpus callosum, cingulum bundle (CGB), and frontal white matter (FWM). LN3 cell density was significantly increased in the CGB, whereas Gal-3 cell density was significantly increased in all regions. Increases in Gal-3 cell density in the FWM were associated with cognitive impairment. In the FWM of old animals, Gal-3-positive microglia were classified by morphological subtype as ramified, hypertrophic, or amoeboid. The densities of hypertrophic and amoeboid microglia significantly correlated with cognitive impairment. Finally, microglia were double-labeled with LN3 and Gal-3 showing that 91% of Gal-3 cells were also LN3 positive, thus expressing an "activated" phenotype. Furthermore, 15% of all double-labeled cells formed phagocytic cups. Overall, these results suggest that microglia become activated in white matter with age where the majority express a phagocytic phenotype. We hypothesize that age-related phagocytic activation of microglia is a response to accumulating myelin pathology. The association of Gal-3 in the FWM with cognitive impairment may reflect regional differences in damage or dysfunction of normal clearance mechanisms.

Chronic curcumin treatment improves spatial working memory but not recognition memory in middle-aged rhesus monkeys.

Studies of both humans and non-human primates have demonstrated that aging is typically characterized by a decline in cognition that can occur as early as the fifth decade of life. Age-related changes in working memory are particularly evident and mediated, in part, by the prefrontal cortex, an area known to evidence age-related changes in myelin that is attributed to inflammation. In recent years, several nutraceuticals, including curcumin, by virtue of their anti-inflammatory and antioxidant effects, have received considerable attention as potential treatments for age-related cognitive decline and inflammation. Accordingly, we assessed for the first time in a non-human primate model of normal aging the efficacy of dietary intervention using the natural phenol curcumin to ameliorate the effects of aging on spatial working and recognition memory. Results revealed that monkeys receiving daily administration of curcumin over 14-18 months demonstrated a greater improvement in performance on repeated administration of a task of spatial working memory compared to monkeys that received a control substance.