Human mesenchymal stem-derived extracellular vesicles improve body growth and motor function following severe spinal cord injury in rat.

BACKGROUND: Spinal cord injury (SCI) in young adults leads to severe sensorimotor disabilities as well as slowing of growth. Systemic pro-inflammatory cytokines are associated with growth failure and muscle wasting. Here we investigated whether intravenous (IV) delivery of small extracellular vesicles (sEVs) derived from human mesenchymal stem/stromal cells (MSC) has therapeutic effects on body growth and motor recovery and can modulate inflammatory cytokines following severe SCI in young adult rats. METHODS: Contusional SCI rats were randomized into three different treatment groups (human and rat MSC-sEVs and a PBS group) on day 7 post-SCI. Functional motor recovery and body growth were assessed weekly until day 70 post-SCI. Trafficking of sEVs after IV infusions in vivo, the uptake of sEVs in vitro, macrophage phenotype at the lesion and cytokine levels at the lesion, liver and systemic circulation were also evaluated. RESULTS: An IV delivery of both human and rat MSC-sEVs improved functional motor recovery after SCI and restored normal body growth in young adult SCI rats, indicating a broad therapeutic benefit of MSC-sEVs and a lack of species specificity for these effects. Human MSC-sEVs were selectively taken up by M2 macrophages in vivo and in vitro, consistent with our previous observations of rat MSC-sEV uptake. Furthermore, the infusion of human or rat MSC-sEVs resulted in an increase in the proportion of M2 macrophages and a decrease in the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-6 at the injury site, as well as a reduction in systemic serum levels of TNF-α and IL-6 and an increase in growth hormone receptors and IGF-1 levels in the liver. CONCLUSIONS: Both human and rat MSC-sEVs promote the recovery of body growth and motor function after SCI in young adult rats possibly via the cytokine modulation of growth-related hormonal pathways. Thus, MSC-sEVs affect both metabolic and neurological deficits in SCI.

Small extracellular vesicles released by infused mesenchymal stromal cells target M2 macrophages and promote TGF-ß upregulation, microvascular stabilization and functional recovery in a rodent model of severe spinal cord injury.

Intravenous (IV) infusion of bone marrow-derived mesenchymal stem/stromal cells (MSCs) stabilizes the blood-spinal cord barrier (BSCB) and improves functional recovery in experimental models of spinal cord injury (SCI). Although IV delivered MSCs do not traffic to the injury site, IV delivered small extracellular vesicles (sEVs) derived from MSCs (MSC-sEVs) do and are taken up by a subset of M2 macrophages. To test whether sEVs released by MSCs are responsible for the therapeutic effects of MSCs, we tracked sEVs produced by IV delivered DiR-labelled MSCs (DiR-MSCs) after transplantation into SCI rats. We found that sEVs were released by MSCs in vivo, trafficked to the injury site, associated specifically with M2 macrophages and co-localized with exosome markers. Furthermore, while a single MSC injection was sufficient to improve locomotor recovery, fractionated dosing of MSC-sEVs over 3 days (F-sEVs) was required to achieve similar therapeutic effects. Infusion of F-sEVs mimicked the effects of single dose MSC infusion on multiple parameters including: increased expression of M2 macrophage markers, upregulation of transforming growth factor-beta (TGF-ß), TGF-ß receptors and tight junction proteins, and reduction in BSCB permeability. These data suggest that release of sEVs by MSCs over time induces a cascade of cellular responses leading to improved functional recovery.

Sodium channel Nav1.6 in sensory neurons contributes to vincristine-induced allodynia.

Vincristine, a widely used chemotherapeutic agent, produces painful peripheral neuropathy. The underlying mechanisms are not well understood. In this study, we investigated whether voltage-gated sodium channels are involved in the development of vincristine-induced neuropathy. We established a mouse model in which repeated systemic vincristine treatment results in the development of significant mechanical allodynia. Histological examinations did not reveal major structural changes at proximal sciatic nerve branches or distal toe nerve fascicles at the vincristine dose used in this study. Immunohistochemical studies and in vivo two-photon imaging confirmed that there is no significant change in density or morphology of intra-epidermal nerve terminals throughout the course of vincristine treatment. These observations suggest that nerve degeneration is not a prerequisite of vincristine-induced mechanical allodynia in this model. We also provided the first detailed characterization of tetrodotoxin-sensitive (TTX-S) and resistant (TTX-R) sodium currents in dorsal root ganglion neurons following vincristine treatment. Accompanying the behavioural hyperalgesia phenotype, voltage-clamp recordings of small and medium dorsal root ganglion neurons from vincristine-treated animals revealed a significant upregulation of TTX-S Na+ current in medium but not small neurons. The increase in TTX-S Na+ current density is likely mediated by Nav1.6, because in the absence of Nav1.6 channels, vincristine failed to alter TTX-S Na+ current density in medium dorsal root ganglion neurons and, importantly, mechanical allodynia was significantly attenuated in conditional Nav1.6 knockout mice. Our data show that TTX-S sodium channel Nav1.6 is involved in the functional changes of dorsal root ganglion neurons following vincristine treatment and it contributes to the maintenance of vincristine-induced mechanical allodynia.

Postirradiation Necrosis after Slow Microvascular Breakdown in the Adult Rat Spinal Cord is Delayed by Minocycline Treatment.

To better understand the spatiotemporal course of radiation-induced central nervous system (CNS) vascular necrosis and assess the therapeutic potential of approaches for protecting against radiation-induced necrosis, adult female Sprague Dawley rats received 40 Gy surface dose centered on the T9 thoracic spinal cord segment. Locomotor function, blood-spinal cord barrier (BSCB) integrity and histology were evaluated throughout the study. No functional symptoms were observed for several months postirradiation. However, a sudden onset of paralysis was observed at approximately 5.5 months postirradiation. The progression rapidly led to total paralysis and death within less than 48 h of symptom onset. Open-field locomotor scores and rotarod motor coordination testing showed no evidence of neurological impairment prior to the onset of overt paralysis. Histological examination revealed minimal changes to the vasculature prior to symptom onset. However, Evans blue dye (EvB) extravasation revealed a progressive deterioration of BSCB integrity, beginning at one week postirradiation, affecting regions well outside of the irradiated area. Minocycline treatment significantly delayed the onset of paralysis. The results of this study indicate that extensive asymptomatic disruption of the blood-CNS barrier may precede onset of vascular breakdown by several months and suggests that minocycline treatment has a therapeutic effect by delaying radiation-induced necrosis after CNS irradiation.

Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord.

In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site [1]. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [2] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSCexos). In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR "hotspots" were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration.

Chronic TNFα Exposure Induces Robust Proliferation of Olfactory Ensheathing Cells, but not Schwann Cells.

TNFα is persistently elevated in many injury and disease conditions. Previous reports of cytotoxicity of TNFα for oligodendrocytes and their progenitors suggest that the poor endogenous remyelination in patients with traumatic injury or multiple sclerosis may be due in part to persistent inflammation. Understanding the effects of inflammatory cytokines on potential cell therapy candidates is therefore important for evaluating the feasibility of their use. In this study, we assessed the effects of long term exposure to TNFα on viability, proliferation, migration and TNFα receptor expression of cultured rat olfactory ensheathing cells (OECs) and Schwann cells (SCs). Although OECs and SCs transplanted into the CNS produce similar myelinating phenotypes, and might be expected to have similar therapeutic uses, we report that they have very different sensitivities to TNFα. OECs exhibited positive proliferative responses to TNFα over a much broader range of concentrations than SCs. Low TNFα concentrations increased proliferation and migration of both OECs and SCs, but SC number declined in the presence of 100 ng/ml or higher concentrations of TNFα. In contrast, OECs exhibited enhanced proliferation even at high TNFα concentrations (up to 1 µg/ml) and showed no evidence of TNF cytotoxicity even at 4 weeks post-treatment. Furthermore, while both OECs and SCs expressed TNFαR1 and TNFαR2, TNFα receptor levels were downregulated in OECs after exposure to100 ng/ml TNFα for 5-7 days, but were either elevated or unchanged in SCs. These results imply that OECs may be a more suitable cell therapy candidate if transplanted into areas with persistent inflammation.

Diffuse and persistent blood-spinal cord barrier disruption after contusive spinal cord injury rapidly recovers following intravenous infusion of bone marrow mesenchymal stem cells.

Intravenous infusion of mesenchymal stem cells (MSCs) has been shown to reduce the severity of experimental spinal cord injury (SCI), but mechanisms are not fully understood. One important consequence of SCI is damage to the microvasculature and disruption of the blood spinal cord barrier (BSCB). In the present study we induced a contusive SCI at T9 in the rat and studied the effects of intravenous MSC infusion on BSCB permeability, microvascular architecture and locomotor recovery over a 10week period. Intravenously delivered MSCs could not be identified in the spinal cord, but distributed primarily to the lungs where they survived for a couple of days. Spatial and temporal changes in BSCB integrity were assessed by intravenous infusions of Evans blue (EvB) with in vivo and ex vivo optical imaging and spectrophotometric quantitation of EvB leakage into the parenchyma. SCI resulted in prolonged BSCB leakage that was most severe at the impact site but disseminated extensively rostral and caudal to the lesion over 6weeks. Contused spinal cords also showed an increase in vessel size, reduced vessel number, dissociation of pericytes from microvessels and decreases in von Willebrand factor (vWF) and endothelial barrier antigen (EBA) expression. In MSC-treated rats, BSCB leakage was reduced, vWF expression was increased and locomotor function improved beginning 1 week post-MSC infusion, i.e., 2weeks post-SCI. These results suggest that intravenously delivered MSCs have important effects on reducing BSCB leakage which could contribute to their therapeutic efficacy.

Intravenous mesenchymal stem cell therapy after recurrent laryngeal nerve injury: a preliminary study.

OBJECTIVES/HYPOTHESIS: Intravenous administration of mesenchymal stem cells (MSCs) has been recently shown to enhance functional recovery after stroke and spinal cord injury. The therapeutic properties of MSCs are attributed to their secretion of a variety of potent antiinflammatory and neurotrophic factors. We hypothesize that intravenous administration of MSCs after recurrent laryngeal nerve (RLN) injury in the rat may enhance functional recovery. STUDY DESIGN: Animal Research. METHODS: Twelve 250-gram Sprague-Dawley rats underwent a controlled crush injury to the left RLN. After confirming postoperative vocal fold immobility, each rat was intravenously infused with either green fluorescent protein-expressing MSCs or control media in a randomized and blinded fashion. Videolaryngoscopy was performed weekly. The laryngoscopy video recordings were reviewed and rated by a fellowship-trained laryngologist who remained blinded to the intervention using a 0 to 3 scale. RESULTS: At 1 week postinjury, the MSC-infused group showed a trend for higher average functional recovery scores compared to the control group (2.2 vs 1.3), but it did not reach statistical significance (P value of 0.06). By 2 weeks, however, both groups exhibited complete return of function. CONCLUSIONS: These pilot data indicate that with complete nerve transection by crush injury of the RLN in rat, there is complete recovery of vocal fold mobility at 2 weeks. At 1 week postinjury, animals receiving intravenous infusion of MSCs showed a trend for greater functional recovery, suggesting a potential beneficial effect of MSCs; however, this did not reach statistical significance. Therefore, no definite conclusions can be drawn from these data and further study is required. LEVEL OF EVIDENCE: N/A.

Olfactory ensheathing cells, but not Schwann cells, proliferate and migrate extensively within moderately X-irradiated juvenile rat brain.

Olfactory ensheathing cells (OECs) and Schwann cells (SCs) share many characteristics, including the ability to promote neuronal repair when transplanted directly into spinal cord lesions, but poor survival and migration when transplanted into intact adult spinal cord. Interestingly, transplanted OECs, but not SCs, migrate extensively within the X-irradiated (40 Gy) adult rat spinal cord, suggesting distinct responses to environmental cues [Lankford et al., (2008) GLIA 56:1664-1678]. In this study, GFP-expressing OECs and SCs were transplanted into juvenile rat brains (hippocampus) subjected to a moderate radiation dose (16 Gy). As in the adult spinal cord, OECs, but not SCs, migrated extensively within the irradiated juvenile rat brain. Unbiased stereology revealed that the number of OECs observed within irradiated rat brains three weeks after transplantation was as much as 20 times greater than the number of cells transplanted, and the cells distributed extensively within the brain. In conjunction with the OEC dispersion, the number of activated microglia in OEC-transplanted irradiated brains was reduced. Unlike in the intact adult spinal cord, both OECs and SCs showed some, but limited, migration within nonirradiated rat brains, suggesting that the developing brain may be a more permissive environment for cell migration than the adult CNS. These results show that OECs display unique migratory, proliferative, and microglia interaction properties as compared with SCs when transplanted into the moderately X-irradiated brain.

Focal experimental autoimmune encephalomyelitis in the Lewis rat induced by immunization with myelin oligodendrocyte glycoprotein and intraspinal injection of vascular endothelial growth factor.

Various models of experimental autoimmune encephalomyelitis (EAE) have led to insights into the pathogenesis and novel therapies for multiple sclerosis. One generalized EAE model uses immunizing the Lewis Rat with myelin oligodendrocyte glycoprotein (MOG) and complete Freund's adjuvant that induces systemic disease and inflammatory lesions at random central nervous system (CNS) locations. These lesions result from a combination of sensitized T cells and pathogenic antibodies gaining access to the CNS to cause an immune assault on myelin-expressing oligodendrocytes. We report a focal and temporal variant of the EAE model that results in immune-mediated demyelination at a predictable time and location. Lewis rats were immunized with the extracellular domain (1-125) of recombinant rat MOG in incomplete Freund's adjuvant (IFA) to induce a clinically silent humoral response. Vascular endothelial growth factor (VEGF) was then microinjected into the spinal cord to induce a transient, focal breakdown of the blood brain barrier (BBB). Clinical signs were apparent within 72 hours and began to resolve by day 21. The histopathology at the site of injection consisted of a focal region containing OX-42(+) cells, phagocytic cells with debris, extensive demyelination, and some lymphocyte infiltration. Neither intraspinal injection of PBS into immunized animals nor VEGF into animals treated with IFA alone resulted in clinical lesions. Thus, a transient, focal opening of the BBB with VEGF in animals with subclinical MOG immunization leads to a discrete inflammatory demyelinating lesion. This model may be useful for the study of transplanted myelin-forming cells in a discrete inflammatory demyelinating lesion.

Convergence of cells from the progenitor fraction of adult olfactory bulb tissue to remyelinating glia in demyelinating spinal cord lesions.

BACKGROUND: Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied. METHODOLOGY/PRINCIPAL FINDINGS: We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings. CONCLUSIONS/SIGNIFICANCE: We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.

Unique in vivo properties of olfactory ensheathing cells that may contribute to neural repair and protection following spinal cord injury.

Olfactory ensheathing cells (OECs) are specialized glial cells that guide olfactory receptor axons from the nasal mucosa into the brain where they make synaptic contacts in the olfactory bulb. While a number of studies have demonstrated that in vivo transplantation of OECs into injured spinal cord results in improved functional outcome, precise cellular mechanisms underlying this improvement are not fully understood. Current thinking is that OECs can encourage axonal regeneration, provide trophic support for injured neurons and for angiogenesis, and remyelinate axons. However, Schwann cell (SC) transplantation also results in significant functional improvement in animal models of spinal cord injury. In culture SCs and OECs share a number of phenotypic properties such as expression of the low affinity NGF receptor (p75). An important area of research has been to distinguish potential differences in the in vivo behavior of OECs and SCs to determine if one cell type may offer greater advantage as a cellular therapeutic candidate. In this review we focus on several unique features of OECs when they are transplanted into the spinal cord.

Transplantation of olfactory ensheathing cells enhances peripheral nerve regeneration after microsurgical nerve repair.

While axonal regeneration is more successful in peripheral nerve than in the central nervous system, it is by no means complete and research to enhance peripheral nerve regeneration is clinically important. Olfactory ensheathing cells (OECs) are known to enhance axonal regeneration and to produce myelin after transplantation. In contrast to Schwann cells their migratory potential and ability to penetrate glial scars is higher. This study evaluated the effect of OEC transplantation on microsurgically repaired sciatic nerves. Rat sciatic nerves were transected followed by microsurgical repair and transplantation of OECs or injection of medium without cells. Twenty-one days later the nerves were removed and prepared for either histology or electrophysiological analysis. Footprint analysis was carried out at 7, 14 and 21 days. The OECs survived and integrated into the repaired nerves as indicated by eGFP-expressing cells aligned with neurofilament identified axons bridging the repair site. Moreover, regenerated axons were myelinated by the transplanted OECs and nodes of Ranvier were formed. Conduction velocity in the OEC transplant group was increased in comparison to the microsurgical repair alone, and improved stepping was observed in the transplant group. These results suggest that presentation of OECs at the time of nerve injury enhances regeneration and improves functional outcome. Even a modest improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.

Induction of parathyroid hormone-related peptide following peripheral nerve injury: role as a modulator of Schwann cell phenotype.

Parathyroid hormone-related peptide (PTHrP) is widely distributed in the rat nervous system, including the peripheral nervous system, where its function is unknown. PTHrP mRNA expression has recently been shown to be significantly elevated following axotomy of sympathetic ganglia, although the role of PTHrP was not investigated. The role of PTHrP in peripheral nerve injury was investigated in this study using the sciatic nerve injury model and dorsal root ganglion (DRG) explant model of nerve regeneration. We find that PTHrP is a constitutively secreted peptide of proliferating Schwann cells and that the PTHrP receptor (PTH1R) mRNA is expressed in isolated DRG and in sciatic nerve. Using the sciatic nerve injury model, we show that PTHrP is significantly upregulated in DRG and in sciatic nerve. In addition, in situ hybridization revealed significant localization of PTHrP mRNA to Schwann cells in the injured sciatic nerve. We also find that PTHrP causes a dramatic increase in the number of Schwann cells that align with and bundle regrowing axons in explants, characteristic of immature, dedifferentiated Schwann cells. In addition to stimulating migration of Schwann cells along the axonal membrane, PTHrP also stimulates migration on a type 1 collagen matrix. Furthermore, treatment of purified Schwann cell cultures with PTHrP results in the rapid phosphorylation of the cAMP response element protein, CREB. We propose that PTHrP acts by promoting the dedifferentiation of Schwann cells, a critical requirement for successful nerve regeneration and an effect consistent with known PTHrP functions in other cellular differentiation programs.

A quantitative morphometric analysis of rat spinal cord remyelination following transplantation of allogenic Schwann cells.

Quantitative morphometric techniques were used to assess the extent and pattern of remyelination produced by transplanting allogenic Schwann cells into demyelinated lesions in adult rat spinal cords. The effects of donor age, prior culturing of donor cells, prior lesioning of donor nerves, and host immunosuppression were evaluated by transplanting suspensions of 30,000 acutely dissociated or cultured Schwann cells from neonatal, young adult, or aged adult rat sciatic nerves into X-irradiation and ethidium bromide-induced demyelinated dorsal column lesions, with or without co-transplantation of neonatal optic nerve astrocytes. Three weeks after transplantation, spinal cords were processed for histological analysis. Under all Schwann cell transplant protocols, large areas containing many Schwann cell-like myelinated axon profiles could be readily observed throughout most of the lesion length. Within these "myelin-rich" regions, the vast majority of detectable axons showed a peripheral-like pattern of myelination. However, interaxonal spacing also increased, resulting in densities of myelinated axons that were more similar to peripheral nerve than intact dorsal columns. Freshly isolated Schwann cells remyelinated more axonal length than cultured Schwann cells, and cells from younger donors remyelinated slightly more axon length than cells from older donors, but all Schwann cell transplant protocols remyelinated tens of thousands of millimeters of axon length and remyelinated axons at similar densities. These results indicate that Schwann cells prepared under a variety of conditions are capable of eliciting remyelination, but that the density of remyelinated axons is much lower than the myelinated axon density in intact spinal cords.