Mesenchymal Stromal Cell–Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils

Abstract

Background and Purpose— Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods— Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV–treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results— sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O 2 ) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O 2 ) but also reduced serum IgG extravasation—a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions— Leukocytes and specifically PMNs contribute to MSC-sEV–induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview— An online visual overview is available for this article.

Homozygous Smpd1 deficiency aggravates brain ischemia/ reperfusion injury by mechanisms involving polymorphonuclear neutrophils, whereas heterozygous Smpd1 deficiency protects against mild focal cerebral ischemia

“…LDF changes were measured during MCAO and up to 15 min after reperfusion onset. Focal cerebral ischemia was induced with an intraluminal filament technique, as previously described [25]. Briefly, a midline neck incision was made, and the left common and external carotid arteries were isolated and ligated.…”

“…On the cresyl violet-stained brain sections, the border between infarcted and non-infarcted tissue was outlined using Image J (National Institutes of Health [NIH], Bethesda, MD, U.S.A.). Infarct area was determined by subtracting the area of the non-lesioned ipsilateral hemisphere from the area of the contralateral hemisphere [25]. In additional cohorts, cryostat sections were collected at millimeter intervals across the forebrain, which were stained with cresyl violet.…”

“…In additional cohorts, cryostat sections were collected at millimeter intervals across the forebrain, which were stained with cresyl violet. On these sections, infarct areas were obtained at all forebrain levels, and infarct volume was calculated by integrating infarct areas across the forebrain [25]. Brain swelling was calculated as area difference of the ipsilateral and the contralateral hemisphere and expressed as percent increase in comparison to the contralateral hemisphere [25].…”

“…Using a commercially available kit (In situ Cell Death Detection Kit; Roche, Basle, Switzerland), DNA-fragmented, that is, irreversibly injured cells were detected in these sections by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) according to the manufacturer's protocol. Sections were evaluated using an inverted microscope (Axio Observer.Z1; Carl Zeiss, Oberkochen, Germany) by counting the density of TUNEL + cells in a region of interest (ROI) measuring 1500 × 1500 µm in the dorsolateral reperfused ischemic striatum [25]. A schematic drawing demonstrating the precise location of this ROI is presented in Suppl.…”

Cell-Free Therapies: Novel Approaches for COVID-19

“…EVs have also been shown to attenuate E. coli and influenza infections (47,48,52), including a mixed swine (H3N2, H1N1) and avian (H9N5, H7N2) influenzainduced lung injury model (48). The beneficial effects of EVs have further been observed in an ischemic stroke model (53,54). Since COVID-19 may be associated with damage to other organs in addition to the lungs and has been associated with ischemic stroke, EVs could be a particularly promising therapy in this context (19).…”