Unpacking the Role of Extracellular Vesicles in Ischemic and Hemorrhagic Stroke: Pathophysiology and Therapeutic Implications.

Stroke is a leading cause of death and disability worldwide. Inflammation and microvascular dysfunction have been associated with brain injury and long-term disability after both ischemic and hemorrhagic stroke. Recent studies have suggested a potential role of extracellular vesicles (EVs) as a link underlying these pathogenic processes. EVs are cell-derived particles enveloped by a lipid bilayer, containing proteins, lipids, and nucleic acids. From a functional standpoint, EVs can facilitate intercellular communication, including across the blood-brain barrier (BBB). Recent advances in EV research have shown a preferential release of EVs from specific cell types in the context of stroke, some of which were associated with increased neuroinflammation, microvascular dysfunction, and neuronal cytotoxicity while others offered a degree of neuroprotection. However, one historic challenge in the studies of EVs in stroke is the lack of consistent definitions and methods to analyze EVs, only recently updated in the MISEV2018 guidelines. Given limitations and complexity in the treatment of stroke, particularly delivery of therapeutics across the BBB, increasing attention has been paid towards manipulating EVs as one vehicle that can permit targeted therapeutic delivery to the central nervous system. These discoveries point towards a future where a better understanding of EVs will advance our knowledge of stroke-associated mechanisms of cerebral and systemic injury and contribute to the development of novel treatments. Here, we review the role that EVs play in ischemic and hemorrhagic stroke.

Bone marrow mesenchymal stromal cells in a 3D system produce higher concentration of extracellular vesicles (EVs) with increased complexity and enhanced neuronal growth properties.

PURPOSE: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems. METHOD: Human bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for ß3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites. RESULTS: The NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (ß3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 µm vs. 1860.5 µm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs (P < 0.05). CONCLUSION: This study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.

VEGF receptor heterodimers and homodimers are differentially expressed in neuronal and endothelial cell types.

PURPOSE: We have previously reported that VEGF-B is more potent than VEGF-A in mediating corneal nerve growth in vitro and in vivo, and this stimulation of nerve growth appears to be different from stimulation of angiogenesis by these same ligands, at least in part due to differences in VEGF receptor activation. VEGF signaling may be modulated by a number of factors including receptor number or the formation of receptor hetero- vs. homodimers. In endothelial cells, VEGF receptor heterodimer (VEGR1/R2) activation after ligand binding and subsequent phosphorylation alters the activation of downstream signaling cascades. However, our understanding of these processes in neuronal cell types remains unclear. The purpose of this study was to identify the presence and distribution of VEGF Receptor-Ligand interactions in neuronal cells as compared to endothelial cells. METHODS: PC12 (rat neuronal cell line), MAEC (mouse aortic endothelial cell line), MVEC (mouse venous endothelial cell line) and HUVEC (human umbilical venous endothelial cell line; control group) were used. Cells were acutely stimulated either with VEGF-A (50 ng/µL) or VEGF-B (50 ng/µL) or "vehicle" (PBS; control group). We also isolated mouse trigeminal ganglion cells from thy1-YFP neurofluorescent mice. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for VEGFR1 and VEGFR2 immunostaining and visualized using confocal fluorescence microscopy and Total Internal Reflection (TIRF) microscopy; (ii) the second group was harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoprecipitation (IP; Thermo Fisher IP kit) and immunoblotting (IB; LI-COR® Systems). Immunoprecipitated proteins were probed either with anti-VEGFR1 or anti-VEGFR2 IgG antibodies to evaluate VEGFR1-R2-heterodimerization; (iii) a third group of cells was also processed for Duolink Proximity Ligation Assay (PLA; Sigma) to assess the presence and distribution of VEGF-receptor homo- and heterodimers in neuronal and endothelial cells. RESULTS: TIRF and fluorescence confocal microscopy revealed the presence of VEGFR1 co-localized with VEGFR2 in endothelial and PC12 neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 further validated the existence of VEGFR1-R2 heterodimers in PC12 neuronal cells. Neuronal cells showed higher levels of VEGFR1-R2 heterodimers as compared to endothelial cells whereas endothelial cells showed higher VEGFR2-R2 homodimers compared to neuronal cells as demonstrated by Duolink PLA. Levels of VEGFR1-R1 homodimers were very low in neuronal and endothelial cells. CONCLUSIONS: Differences in VEGF Receptor homo- and heterodimer distribution may explain the differential role of VEGF ligands in neuronal versus endothelial cell types. This may in turn influence VEGF activity and regulation of neuronal cell homeostasis.

Combinatorial therapy with immunosuppressive, immunomodulatory and tear substitute eyedrops ("Triple Play") in Recalcitrant Immunological Ocular Surface Diseases.

PURPOSE: The current paradigm for therapy of recalcitrant ocular surface diseases (OSD) consists of a sequential, step-up treatment approach. A combinatorial topical therapy (anti-inflammatory/immunosuppressive [steroid] with immunomodulatory [pooled human immune globulin] and tear substitute [serum]) that simultaneously targets several immunological pathways may be more efficacious. This report evaluates if the combinatorial therapy resulted in clinical benefit in patients with recalcitrant OSD. METHODS: We performed a retrospective case study of patients receiving topical, preservative-free, compounded formulations of steroids, pooled human immune globulin, and serum tears. Outcome measures included visual acuity, ocular surface disease index (OSDI), ocular discomfort score, subjective global assessment (SGA), corneal staining, conjunctival redness, and slit lamp photographs. RESULTS: Patients consisted of one male and 11 females ranging in age from 27 to 87 years old. Pathologies included ocular graft-versus-host disease (n = 4), Sjögren's syndrome (n = 3), ocular cicatricial pemphigoid (n = 1), pemphigus vulgaris (n = 1), peripheral ulcerative keratitis (n = 1), Stevens-Johnson syndrome (n = 1), and giant papillary conjunctivitis (n = 1). All patients were "improved" or "much improved" on SGA after combinatorial therapy. There was a clinically meaningful reduction in OSDI, ocular discomfort, corneal staining, and conjunctival injection. Additionally, three patients had improvement in their visual acuity (one from 20/400 to 20/20). Adverse effects included increased intraocular pressure in two patients, presumably due to topical steroid use. CONCLUSIONS: Combinatorial therapy provides clinical benefit by reducing the symptoms and signs in recalcitrant OSD. Our study provides the rationale for performing prospective clinical trials to evaluate the efficacy of combinatorial therapy for treating recalcitrant OSD.

The Limbal Niche and Regenerative Strategies.

The protective function and transparency provided by the corneal epithelium are dependent on and maintained by the regenerative capacity of limbal epithelial stem cells (LESCs). These LESCs are supported by the limbal niche, a specialized microenvironment consisting of cellular and non-cellular components. Disruption of the limbal niche, primarily from injuries or inflammatory processes, can negatively impact the regenerative ability of LESCs. Limbal stem cell deficiency (LSCD) directly hampers the regenerative ability of the corneal epithelium and allows the conjunctival epithelium to invade the cornea, which results in severe visual impairment. Treatment involves restoring the LESC population and functionality; however, few clinically practiced therapies currently exist. This review outlines the current understanding of the limbal niche, its pathology and the emerging approaches targeted at restoring the limbal niche. Most emerging approaches are in developmental phases but show promise for treating LSCD and accelerating corneal regeneration. Specifically, we examine cell-based therapies, bio-active extracellular matrices and soluble factor therapies in considerable depth.

Systemic Immune-Inflammation Index Predicts Delayed Cerebral Vasospasm After Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Delayed cerebral vasospasm is a feared complication of aneurysmal subarachnoid hemorrhage (SAH). OBJECTIVE: To investigate the relationship of systemic inflammation, measured using the systemic immune-inflammation (SII) index, with delayed angiographic or sonographic vasospasm. We hypothesize that early elevations in SII index serve as an independent predictor of vasospasm. METHODS: We retrospectively reviewed the medical records of 289 SAH patients for angiographic or sonographic evidence of delayed cerebral vasospasm. SII index [(neutrophils × platelets/lymphocytes)/1000] was calculated from laboratory data at admission and dichotomized based on whether or not the patient developed vasospasm. Multivariable logistic regression and receiver operating characteristic (ROC) analysis were performed to determine the ability of SII index to predict the development of vasospasm. RESULTS: A total of 246 patients were included in our study, of which 166 (67.5%) developed angiographic or sonographic evidence of cerebral vasospasm. Admission SII index was elevated for SAH in patients with vasospasm compared to those without (P < .001). In univariate logistic regression, leukocytes, neutrophils, lymphocytes, neutrophil-lymphocyte ratio (NLR), and SII index were associated with vasospasm. After adjustment for age, aneurysm location, diabetes mellitus, hyperlipidemia, and modified Fisher scale, SII index remained an independent predictor of vasospasm (odds ratio 1.386, P = .003). ROC analysis revealed that SII index accurately distinguished between patients who develop vasospasm vs those who do not (area under the curve = 0.767, P < .001). CONCLUSION: Early elevation in SII index can independently predict the development of delayed cerebral vasospasm in aneurysmal SAH.

Inflammasome Caspase-1 Activity is Elevated in Cerebrospinal Fluid After Aneurysmal Subarachnoid Hemorrhage and Predicts Functional Outcome.

BACKGROUND/OBJECTIVE: Subarachnoid hemorrhage (SAH) is a devastating neurological injury, further complicated by few available methods to objectively predict outcomes. With the recent shift in focus to neuroinflammation as a potential cause of adverse outcomes following SAH, we investigated the inflammasome-derived enzyme, caspase-1, as a potential biomarker for poor functional outcome. METHODS: SAH patients were recruited from a regional stroke referral center. Cerebrospinal fluid (CSF) samples from 18 SAH subjects were collected via an external ventricular drain and obtained as close as possible to admission (within 72 h). For control subjects, we collected CSF from 9 patients undergoing lumbar puncture with normal CSF. Caspase-1 activity was measured using commercially available luminescence assays. SAH subjects were categorized at hospital discharge into those with good outcomes (Glasgow Outcome Scale, GOS, of 4-5) and poor outcomes (GOS of 1-3). RESULTS: CSF analysis demonstrated a nearly seven-fold increase in caspase-1 activity in SAH patients compared to controls (p < 0.0001). Within the SAH group, 10 patients (55.6%) had good outcomes and 8 patients (44.4%) had poor outcomes. Mean caspase-1 activity in the poor outcome group was approximately three-times higher than the good outcome group (p = 0.001). Caspase-1 activity was significantly correlated with GOS score (r = - 0.705, p = 0.001). Receiver operating characteristic curve analysis showed that caspase-1 activity can accurately differentiate between patients with good versus poor functional outcome (area under the curve 0.944, p = 0.002). CONCLUSIONS: Inflammasome-derived caspase-1 activity is elevated in the CSF of SAH patients compared to controls and higher levels correlate with worse functional outcome.

Resonance with subthreshold oscillatory drive organizes activity and optimizes learning in neural networks.

Network oscillations across and within brain areas are critical for learning and performance of memory tasks. While a large amount of work has focused on the generation of neural oscillations, their effect on neuronal populations' spiking activity and information encoding is less known. Here, we use computational modeling to demonstrate that a shift in resonance responses can interact with oscillating input to ensure that networks of neurons properly encode new information represented in external inputs to the weights of recurrent synaptic connections. Using a neuronal network model, we find that due to an input current-dependent shift in their resonance response, individual neurons in a network will arrange their phases of firing to represent varying strengths of their respective inputs. As networks encode information, neurons fire more synchronously, and this effect limits the extent to which further "learning" (in the form of changes in synaptic strength) can occur. We also demonstrate that sequential patterns of neuronal firing can be accurately stored in the network; these sequences are later reproduced without external input (in the context of subthreshold oscillations) in both the forward and reverse directions (as has been observed following learning in vivo). To test whether a similar mechanism could act in vivo, we show that periodic stimulation of hippocampal neurons coordinates network activity and functional connectivity in a frequency-dependent manner. We conclude that resonance with subthreshold oscillations provides a plausible network-level mechanism to accurately encode and retrieve information without overstrengthening connections between neurons.