Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) have been proposed to be a key mechanistic link in the therapeutic efficacy of cells in response to cellular injuries through paracrine effects. We hypothesize that inflammatory stimulation of MSCs results in the release of EVs that have greater anti-inflammatory effects. The present study evaluates the immunomodulatory abilities of EVs derived from inflammation-stimulated and naive MSCs (MSCEv 1 and MSCEv, respectively) isolated using a current Good Manufacturing Practice-compliant tangential flow filtration system. Detailed characterization of both EVs revealed differences in protein composition, cytokine profiles, and RNA content, despite similarities in size and expression of common surface markers. MSCEv 1 further attenuated release of pro-inflammatory cytokines in vitro when compared to MSCEv, with a distinctly different pattern of EV-uptake by activated primary leukocyte subpopulations. The efficacy of EVs was partially attributed to COX2/PGE 2 expression. The present study demonstrates that inflammatory stimulation of MSCs renders release of EVs that have enhanced anti-inflammatory properties partially due to COX2/PGE 2 pathway alteration. STEM CELLS 2018;36:79-90 SIGNIFICANCE STATEMENTPrevious work has identified mesenchymal stromal cell-derived extracellular vesicles (MSCEv) as mediators of cell-cell communication and effectors of cellular/tissue change. This study isolated MSCEv using a clinically propitious filtration system after stimulation with inflammatory cytokines, characterized their composition, and evaluated their effect on inflammation, along with their potential mechanism of action and interaction with potential target cells. This study identified important compositional differences between control and stimulated MSCEv in cytokine and RNA content. Furthermore, stimulated MSCEv attenuate TNF-a and IFN-g release from activated splenocytes compared to standard MSCEv (and liposomal controls). The nature of MSCEv interaction with cells likely involves cellular internalization, so this study fluorescently labeled MSCEv prior to coculture with activated leukocytes to determine changes in uptake activity in response to several antigens. These studies demonstrate a specific anti-inflammatory, MSCEvmediated response and the capacity to change efficacy in response to inflammatory cues, creating the foundation for enhancing the efficacy of translational efforts using MSCEv for targeting inflammatory injuries and diseases. This represents a new paradigm for generation of extracellular vesicles targeting specific pathologies.
Corticotropin-releasing factor receptor CRF1 has been implicated in the neurobiological mechanisms of anxiety and depression. The amygdala plays an important role in affective states and disorders such as anxiety and depression. The amygdala is also emerging as a neural substrate of pain affect. However, the involvement of the amygdala in the interaction of pain and anxiety remains to be determined. This study tested the hypothesis that CRF1 receptors in the amygdala are critically involved in pain-related anxiety. Anxiety-like behavior was determined in adult male rats using the elevated plus maze (EPM) test. The open-arm preference (ratio of open arm entries to the total number of entries) was measured. Nocifensive behavior was assessed by measuring hindlimb withdrawal thresholds for noxious mechanical stimulation of the knee. Measurements were made in normal rats and in rats with arthritis induced in one knee by intraarticular injections of kaolin/carrageenan. A selective CRF1 receptor antagonist (NBI27914) or vehicle was administered systemically (i.p.) or into the central nucleus of the amygdala (CeA, by microdialysis). The arthritis group showed a decreased preference for the open arms in the EPM and decreased hindlimb withdrawal thresholds. Systemic or intraamygdalar (into the CeA) administration of NBI27914, but not vehicle, inhibited anxiety-like behavior and nocifensive pain responses, nearly reversing the arthritis pain-related changes. This study shows for the first time that CRF1 receptors in the amygdala contribute critically to pain-related anxiety-like behavior and nocifensive responses in a model of arthritic pain. The results are a direct demonstration that the clinically welldocumented relationship between pain and anxiety involves the amygdala.
No current clinical intervention can alter the course of acute spinal cord injury (SCI), or appreciably improve neurological outcome. Mesenchymal stromal cells (MSCs) have been shown to modulate the injury sequelae of SCI largely via paracrine effects, although the mechanisms remain incompletely understood. One potential modality is through secretion of extracellular vesicles (EVs). In this study, we investigate whether systemic administration of EVs isolated from human MSCs (MSCEv) has the potential to be efficacious as an alternative to cell-based therapy for SCI. Additionally, we investigate whether EVs isolated from human MSCs stimulated with pro-inflammatory cytokines have enhanced anti-inflammatory effects when administered after SCI. Immunohistochemistry supported the quantitative analysis, demonstrating a diminished inflammatory response with apparent astrocyte and microglia disorganization in cord tissue up to 10 mm caudal to the injury site. Locomotor recovery scores showed significant improvement among animals treated with MSCEv. Significant increases in mechanical sensitivity threshold were observed in animals treated with EVs from either naïve MSC (MSCEvwt) or stimulated MSC (MSCEv+), with a statistically significant increase in threshold for MSCEv+-treated animals when compared to those that received MSCEvwt. In conclusion, these data show that treatment of acute SCI with extracellular vesicles derived from human MSCs attenuates neuroinflammation and improves functional recovery.
Surgical drainage of intracranial hematomas is often required within the first four hours after traumatic brain injury (TBI) to avoid death or severe disability. Although CT and MRI permit hematoma diagnosis, they can be used only at a major health-care facility. This delays hematoma diagnosis and therapy. We proposed to use an optoacoustic technique for rapid, noninvasive diagnosis of hematomas. In this study we developed a near-infrared OPO-based optoacoustic system for hematoma diagnosis and cerebral venous blood oxygenation monitoring in rats. A specially-designed blast device was used to inflict TBI in anesthetized rats. Optoacoustic signals were recorded from the superior sagittal sinus and hematomas that allowed for measurements of their oxygenations. These results indicate that the optoacoustic technique may be used for early diagnosis of hematomas and may provide important information for improving outcomes in patients with TBI.
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