Intranasally Administered Human MSC-Derived Extracellular Vesicles Pervasively Incorporate into Neurons and Microglia in both Intact and Status Epilepticus Injured Forebrain

Abstract

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (hMSCs) have great promise as biologics to treat neurological and neurodegenerative conditions due to their robust antiinflammatory and neuroprotective properties. Besides, intranasal (IN) administration of EVs has caught much attention because the procedure is noninvasive, amenable for repetitive dispensation, and leads to a quick penetration of EVs into multiple regions of the forebrain. Nonetheless, it is unknown whether brain injury-induced signals are essential for the entry of IN-administered EVs into different brain regions. Therefore, in this study, we investigated the distribution of IN-administered hMSC-derived EVs into neurons and microglia in the intact and status epilepticus (SE) injured rat forebrain. Ten billion EVs labeled with PKH26 were dispensed unilaterally into the left nostril of naïve rats, and rats that experienced two hours of kainate-induced SE. Six hours later, PKH26 + EVs were quantified from multiple forebrain regions using serial brain sections processed for different neural cell markers and confocal microscopy. Remarkably, EVs were seen bilaterally in virtually all regions of intact and SE-injured forebrain. The percentage of neurons incorporating EVs were comparable for most forebrain regions. However, in animals that underwent SE, a higher percentage of neurons incorporated EVs in the hippocampal CA1 subfield and the entorhinal cortex, the regions that typically display neurodegeneration after SE. In contrast, the incorporation of EVs by microglia was highly comparable in every region of the forebrain measured. Thus, unilateral IN administration of EVs is efficient for delivering EVs bilaterally into neurons and microglia in multiple regions in the intact or injured forebrain. Furthermore, incorporation of EVs by neurons is higher in areas of brain injury, implying that injury-related signals likely play a role in targeting of EVs into neurons, which may be beneficial for EV therapy in various neurodegenerative conditions including traumatic brain injury, stroke, multiple sclerosis, and Alzheimer’s disease.

Intranasal delivery of mesenchymal stem cell-derived extracellular vesicles exerts immunomodulatory and neuroprotective effects in a 3xTg model of Alzheimer's disease

“…IN administration of EVs has been tested in neurological disorders including Parkinson's disease 82 and status epilepticus (SE), both in pilocarpine‐induced 50 or kainate‐induced 83 models of SE, lipopolysaccharide (LPS)‐induced brain inflammation model, experimental autoimmune encephalomyelitis, GL26 tumor model, 84 and the autism BTBR T+tf/J (BTBR) mice model 85 . However, except for the LPS‐acute inflammatory model, 84 all these studies have exploited chronic EV treatment, with mice being sacrificed at the end of the treatment.…”

Extracellular vesicles from human iPSC‐derived neural stem cells: miRNA and protein signatures, and anti‐inflammatory and neurogenic properties

“…The rats and mice (n = 2/species) were moderately anesthetized with a cocktail of ketamine, xylazine and acepromazine, and their nostrils were treated with 10 µl of hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) to enhance the permeability of the nasal mucous membrane. Thirty minutes later, labelled EVs were administered (150 × 10 9 [~75 µg]/rat and 25 × 10 9 [~12.5 µg]/mouse), as detailed in our recent report [22]. Six hours later, the animals were perfused, the brain tissues were processed for cryostat sectioning, and thirty-micrometre-thick coronal sections through the entire forebrain, midbrain and hindbrain, were collected serially.…”

“…Six hours later, the animals were perfused, the brain tissues were processed for cryostat sectioning, and thirty-micrometre-thick coronal sections through the entire forebrain, midbrain and hindbrain, were collected serially. Serial sections through the entire brain were selected and processed for immunofluorescent staining using appropriate primary antibodies against NeuN (1:1000; Millipore), IBA-1 (1:1000; Abcam), GFAP (1:3000; Dako, Glostrup, Denmark), S-100β (1:1000; Sigma) and calbindin (1:500; Sigma), as described in our previous report [22]. The sections were examined using Z-section analysis in a confocal microscope for the distribution of EVs in different cell types (neurons, microglia and astrocytes) in multiple regions of the brain.…”

“…Indeed, several studies have reported the therapeutic efficacy of EVs derived from various stem cells in CNS repair and regeneration after injury or disease [17][18][19][20][21]. Moreover, EVs are amenable for repeated, noninvasive dispensation as an autologous or allogeneic off-the-shelf product for treating neurological diseases because they can quickly permeate the entire brain following an intranasal administration [22].…”

The Interstitial System of the Brain in Health and Disease

“…A similar process likely also aids the quick entry of nanosized EVs into the brain when administered intranasally. Recent studies have shown that intranasally administered stem cell-derived EVs permeate the entire forebrain and incorporate into different neural cells within 6 hours [49,50]. Such rapid targeting by EVs is likely due to their quick entry into the subarachnoid space through perineurial spaces around olfactory nerves passing through the cribriform plate and subsequent transportation through CSF flow into the ISF.…”