Survival Of Oligodendrocyte Progenitor Cells Research Articles

Grafted human-induced pluripotent stem cells-derived oligodendrocyte progenitor cells combined with human umbilical vein endothelial cells contribute to functional recovery following spinal cord injury

BackgroundSpinal cord injury (SCI) is a devastating disease that causes extensive damage to oligodendrocytes and neurons leading to demyelination and axonal degeneration. In this study, we co-transplanted cell grafts containing oligodendrocyte progenitor cells (OPCs) derived from human-induced pluripotent stem cells (iPSCs) combined with human umbilical vein endothelial cells (HUVECs), which were reported to promote OPCs survival and migration, into rat contusion models to promote functional recovery after SCI.MethodsOPCs were derived from iPSCs and identified by immunofluorescence at different time points. Functional assays in vitro were performed to evaluate the effect of HUVECs on the proliferation, migration, and survival of OPCs by co-culture and migration assay, as well as on the neuronal axonal growth. A combination of OPCs and HUVECs was transplanted into the rat contusive model. Upon 8 weeks, immunofluorescence staining was performed to test the safety of transplanted cells and to observe the neuronal repairment, myelination, and neural circuit reconstruction at the injured area; also, the functional recovery was assessed by Basso, Beattie, and Bresnahan open-field scale, Ladder climb, SEP, and MEP. Furthermore, the effect of HUVECs on grafts was also determined in vivo.ResultsData showed that HUVECs promote the proliferation, migration, and survival of OPCs both in vitro and in vivo. Furthermore, 8 weeks upon engraftment, the rats with OPCs and HUVECs co-transplantation noticeably facilitated remyelination, enhanced functional connection between the grafts and the host and promoted functional recovery. In addition, compared with the OPCs-alone transplantation, the co-transplantation generated more sensory neurons at the lesion border and significantly improved the sensory functional recovery.ConclusionsOur study demonstrates that transplantation of OPCs combined with HUVECs significantly enhances both motor and sensory functional recovery after SCI. No significance was observed between OPCs combined with HUVECs group and OPCs-alone group in motor function recovery, while the sensory function recovery was significantly promoted in OPCs combined with HUVECs groups compared with the other two groups. These findings provide novel insights into the field of SCI research.

Administration of the GLP-1 receptor agonist exenatide in rats improves functional recovery after spinal cord injury by reducing endoplasmic reticulum stress

After spinal cord injury (SCI), endoplasmic reticulum (ER) stress has been reported to be an integral part of the secondary injury process that causes apoptosis of glial cells, leading to remyelination failure. This report focuses on exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist widely used to treat diabetes, as a potential agent to improve functional outcome after SCI by improving the ER stress response. Exenatide administered subcutaneously immediately after injury and 7 days later in a rat model of moderate contusive SCI revealed significant improvement in hindlimb function without any hypoglycemia. Changes in the expression of glucose regulatory protein 78 (GRP78), an endoplasmic reticulum chaperone that protects against ER stress, and C/EBP homologous transcription factor protein (CHOP), a pro-apoptotic transcription factor in the apoptosis pathway were examined as indices of ER stress. We found that administration of exenatide after SCI suppressed CHOP while increasing GRP78 in the injured spinal cord, leading to a significant decrease in tissue damage and a significant increase in oligodendrocyte progenitor cell survival. This study suggests that administration of exenatide after SCI decreases ER stress and improves functional recovery without any apparent side-effects.

Photobiomodulation attenuates oligodendrocyte dysfunction and prevents adverse neurological consequences in a rat model of early life adversity.

Rationale: Adverse experiences in early life including abuse, trauma and neglect, have been linked to poor physical and mental health outcomes. Emerging evidence implies that those who experienced early life adversity (ELA) are more likely to develop cognitive dysfunction and depressive-like symptoms in adulthood. The molecular mechanisms responsible for the negative consequences of ELA, however, remain unclear. In the absence of effective management options, anticipatory guidance is the mainstay of ELA prevention. Furthermore, there is no available treatment that prevents or alleviates the neurologic sequelae of ELA, especially traumatic stress. Hence, the present study aims to investigate the mechanisms for these associations and evaluate whether photobiomodulation (PBM), a non-invasive therapeutic procedure, can prevent the negative cognitive and behavioral manifestations of ELA in later life. Methods: ELA was induced by repeated inescapable electric foot shock of rats from postnatal day 21 to 26. On the day immediately following the last foot shock, 2-min daily PBM treatment was applied transcranially for 7 consecutive days. Cognitive dysfunction and depression-like behaviors were measured by a battery of behavioral tests in adulthood. Subsequently, oligodendrocyte progenitor cells (OPCs) differentiation, the proliferation and apoptosis of oligodendrocyte lineage cells (OLs), mature oligodendrocyte, myelinating oligodendrocyte, the level of oxidative damage, reactive oxygen species (ROS) and total antioxidant capacity were measured and analyzed using immunofluorescence staining, capillary-based immunoassay (ProteinSimple®) and antioxidant assay kit. Results: The rats exposed to ELA exhibited obvious oligodendrocyte dysfunction, including a reduction in OPCs differentiation, diminished generation and survival of OLs, decreased OLs, and decreased matured oligodendrocyte. Furthermore, a deficit in myelinating oligodendrocytes was observed, in conjunction with an imbalance in redox homeostasis and accumulated oxidative damage. These alternations were concomitant with cognitive dysfunction and depression-like behaviors. Importantly, we found that early PBM treatment largely prevented these pathologies and reversed the neurologic sequelae resulting from ELA. Conclusions: Collectively, these findings provide new insights into the mechanism by which ELA affects neurological outcomes. Moreover, our findings support that PBM may be a promising strategy to prevent ELA-induced neurologic sequelae that develops later in life.

Genetically modified macrophages accelerate myelin repair.

Preventing neurodegeneration‐associated disability progression in patients with multiple sclerosis (MS) remains an unmet therapeutic need. As remyelination prevents axonal degeneration, promoting this process in patients might enhance neuroprotection. In demyelinating mouse lesions, local overexpression of semaphorin 3F (Sema3F), an oligodendrocyte progenitor cell (OPC) attractant, increases remyelination. However, molecular targeting to MS lesions is a challenge. A clinically relevant paradigm for delivering Sema3F to demyelinating lesions could be to use blood‐derived macrophages as vehicles. Thus, we chose transplantation of genetically modified hematopoietic stem cells (HSCs) as means of obtaining chimeric mice with circulating Sema3F‐overexpressing monocytes. We demonstrated that Sema3F‐transduced HSCs stimulate OPC migration in a neuropilin 2 (Nrp2, Sema3F receptor)‐dependent fashion, which was conserved in middle‐aged OPCs. While demyelinating lesions induced in mice with Sema3F‐expressing blood cells showed no changes in inflammation and OPC survival, OPC recruitment was enhanced which accelerated the onset of remyelination. Our results provide a proof of concept that blood cells, particularly monocytes/macrophages, can be used to deliver pro‐remyelinating agents “at the right time and place,” suggesting novel means for remyelination‐promoting strategies in MS.

376 Hemoglobin Disrupts Microglial Function in the Developing Brain

INTRODUCTION: Intraventricular hemorrhage of prematurity (IVH) interrupts normal white matter development. During normal brain development, microglia play an important role in myelination by producing insulin-like growth factor 1 (IGF-1) which is necessary for oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes. IGF-1 is produced by a subpopulation of microglia that express the antigen CD11c, and these microglia are enriched during early brain development. METHODS: A cell culture system of isolated microglia was used for in vitro studies. A rat model of neonatal IVH was used for in vivo studies. Magnetic bead sorting was used to isolate CD11c+ microglia. Real-time PCR was used to quantify IGF-1 expression. ELISA was used to measure IGF-1 and cytokine production. Immunohistochemistry was used to quantify white matter and label OPCs, microglia, and CD11c. RESULTS: Hemoglobin induced a greater degree of TNFa production compared to an equivalent dose of whole blood. in vitro, hemoglobin reduced the percentage of microglia that expressed CD11c, and reduced the IGF-1production measured by ELISA. In an animal model of neonatal IVH, where hemoglobin injection reduces white matter development but not OPC survival, CD11c+ cells decreased their IGF-1 production as measured by real-time PCR. CONCLUSION: Hemoglobin is a potent pro-inflammatory stimulus for microglia in the developing brain. Hemoglobin induces classically proinflammatory microglial activation, with production of cytokines and reduction of IGF-1 production. CD11c+ microglia, the cells that provide the IGF-1 critical for myelination, reduce IGF-1 production in vivo when exposed to hemoglobin. This loss of microglial IGF-1 may be the reason OPCs fail to mature and myelinate effectively. Replacing IGF-1 maybe a therapeutic strategy for neonatal IVH.

Lanthionine Ketimine Ethyl Ester Accelerates Remyelination in a Mouse Model of Multiple Sclerosis.

Although over 20 disease modifying therapies are approved to treat Multiple Sclerosis (MS), these do not increase remyelination of demyelinated axons or mitigate axon damage. Previous studies showed that lanthionine ketenamine ethyl ester (LKE) reduces clinical signs in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and increased maturation of oligodendrocyte (OL) progenitor cells (OPCs) in vitro. In the current study, we used the cuprizone (CPZ) demyelination model of MS to test if LKE could increase remyelination. The corpus callosum (CC) and somatosensory cortex was examined by immunohistochemistry (IHC), electron microscopy and for mRNA expression changes in mice provided 5 weeks of CPZ diet followed by 2 weeks of normal diet in the presence of LKE or vehicle. A significant increase in the number of myelinated axons, and increased myelin thickness was observed in the CC of LKE-treated groups compared to vehicle-treated groups. LKE also increased myelin basic protein and proteolipid protein expression in the CC and cortex, and increased the number of mature OLs in the cortex. In contrast, LKE did not increase the percentage of proliferating OPCs suggesting effects on OPC survival and differentiation but not proliferation. The effects of LKE on OL maturation and remyelination were supported by similar changes in their relative mRNA levels. Interestingly, LKE did not have significant effects on GFAP or Iba1 immunostaining or mRNA levels. These findings suggest that remyelinating actions of LKE can potentially be formulated to induce remyelination in neurological diseases associated with demyelination including MS.

Azithromycin Protects Oligodendrocyte Progenitor Cells against Lipopolysaccharide-Activated Microglia-Induced Damage

Oligodendrocyte progenitor cells (OPC) are the primary cellular targets of brain white matter injury (WMI) in very low-birth weight (VLBW) infants. Microglia plays a significant role in inflammation-induced WMI. Our previous study showed that lipopolysaccharide (LPS)-induced OPC damage is mediated by activated microglia in vitro. We hypothesized that azithromycin (AZ) could protect OPCs against LPS-induced cytotoxicity by blocking microglial activation. Highly enriched primary rat microglia and OPCs were treated with LPS. There were 4 groups: control, LPS + Veh, AZ, and LPS + AZ. Microglia conditioned medium (MCM) was used to determine inflammatory cytokines by enzyme-linked immunosorbent assay or subsequent treatment of OPCs. We found that AZ significantly suppressed TNF-α, IL-1β, and IL-6 in LPS+Veh-treated–microglial MCM and blocked microglial nuclear factor-κB p65 nuclear translocation. AZ prevented LPS-MCM-induced OPC death and improved OPC survival as measured by activated caspase-3 immunostaining and XTT assay, respectively. AZ ameliorated LPS-MCM-induced differentiation arrest and myelin basic protein deficit in oligodendrocytes. Our data suggest that AZ is a potent inhibitor for microglia activation and may hold the therapeutic potential for WMI in VLBW infants.

Survival control of oligodendrocyte progenitor cells requires the transcription factor 4 during olfactory bulb development

A proper number of oligodendrocytes in the nerve system is essential for neuronal functions. In the olfactory bulb (OB), enriched oligodendrocytes are crucial for olfactory information processing. However, how the precise number of oligodendrocytes in the OB is regulated remains elusive. Here we identified that the transcription factor 4 (Tcf4)-mediated cell death is essential for generating an appropriate number of oligodendrocyte progenitor cells (OPCs) and thereby oligodendrocytes in the OB. We showed that Nkx2.1-positive progenitors in the medial ganglionic eminence (MGE) and anterior entopeduncular area (AEP) provide the first source of OPCs in the OB. Conditional depletion of Tcf4 leads to an increase of OPCs in the OB, which is mediated by the suppression of programmed cell death. Furthermore, we showed that Tcf4 mediated OPC survival is cell-autonomous by transplantation assay. Mechanistically, we identified Bax/Bak as a potential key pathway to promote OPC elimination during OB development. Depletion of Bax/Bak in Nkx2.1 lineage results in an increase of OPCs in the OB. Mutations in TCF4 causes Pitt-Hopkins syndrome, a severe neurodevelopmental disorder. Thus, our findings reveal an important intrinsic mechanism underlying the survival control of OPCs in the OB and provide new insights into the pathogenesis of Pitt–Hopkins syndrome.

Effect of fluoxetine on proliferation and/or survival of microglia and oligodendrocyte progenitor cells in the fornix and corpus callosum of the mouse brain.

Fluoxetine is one of the most widely prescribed antidepressants and a selective inhibitor of presynaptic 5-HT transporters. The fornix is the commissural and projection fiber that transmits signals from the hippocampus to other parts of the brain and opposite site of hippocampus. The corpus callosum (CC) is the largest of the commissural fibers that link the cerebral cortex of the left and right cerebral hemispheres. These brain regions play pivotal roles in cognitive functions, and functional abnormalities in these regions have been implicated in the development of various brain diseases. The purpose of the present study was to investigate the effects of fluoxetine on the proliferation and/or survival of microglia and oligodendrocyte progenitor cells (OPCs) in the fornix and CC, the white matter connecting cortical-limbic system, of the adult mouse brain. The effects of fluoxetine on the proliferation and/or survival of microglia and OPCs were examined in lipopolysaccharide (LPS)-treated and normal mice. Proliferating cells were detected in mice that drank water containing the thymidine analog, bromodeoxyuridine (BrdU), using immunohistochemistry. Fluoxetine significantly attenuated LPS-induced increases in the number of BrdU-labeled microglia and morphological activation from the ramified to ameboid shape, and decreased the number of BrdU-labeled OPCs under basal conditions. The present results indicate that fluoxetine exerts inhibitory effects on LPS-induced increases in the proliferation and/or survival and morphological activation of microglia and basal proliferation and/or survival of OPCs in the fornix and CC of adult mice.

Oligogenesis in the "oligovascular unit" involves PI3K/AKT/mTOR signaling in hypoxic-ischemic neonatal mice

The "oligovascular unit" is a dynamic structural complex composed of endothelial cells (ECs) and oligodendrocyte progenitor cells (OPCs)/oligodendrocytes. By improving the microenvironment of OPCs in the "oligovascular unit" and promoting the proliferation and differentiation of OPCs, both myelination and white matter injury can be repaired. However, it is unclear what characteristic changes occur in the microenvironment of the "oligovascular unit" after preterm white matter injury (PWMI). Here, we demonstrate the changes in the "oligovascular unit" induced by hypoxia-ischemia (HI) and its underlying mechanism in PWMI mice. White matter injury and inhibited production of myelin from OPCs were observed by histopathological staining in HI neonatal mice. We further observed that the proliferation of OPCs and angiogenesis were increased after HI, which is considered the response of the body and cells to HI. HI-induced oligogenesis occurs around the vessels, indicating that "oligovascular units" exist and promote the proliferation and differentiation of OPCs after HI in the short term. We also determined that angiogenesis and oligogenesis induced by HI in the white matter are related to the PI3K/AKT/mTOR pathway. Furthermore, the myelin sheaths were shown to be disordered on the side of the surgery, and the myelin-dense layer was poorly developed at P14 and P28. Different degrees of damage to the vascular ECs and basement membrane on the surgical side were detected beginning at P4, indicating that EC injury is an early phenomenon that subsequently affects oligogenesis. Taken together, our findings indicate that the proliferation of OPCs and angiogenesis in white matter are increased in the early stage of HI involving PI3K/AKT/mTOR pathway activation. Promoting vascular endothelial function and angiogenesis may increase the proliferation and survival of OPCs via the "oligovascular unit," which suggests a potential method to repair injured white matter in the early stage of PWMI.

Autophagy is essential for oligodendrocyte differentiation, survival, and proper myelination.

Deficient myelination, the spiral wrapping of highly specialized membrane around axons, causes severe neurological disorders. Maturation of oligodendrocyte progenitor cells (OPC) to myelinating oligodendrocytes (OL), the sole providers of central nervous system (CNS) myelin, is tightly regulated and involves extensive morphological changes. Here, we present evidence that autophagy, the targeted isolation of cytoplasm and organelles by the double-membrane autophagosome for lysosomal degradation, is essential for OPC/OL differentiation, survival, and proper myelin development. A marked increase in autophagic activity coincides with OL differentiation, with OL processes having the greatest increase in autophagic flux. Multiple lines of evidence indicate that autophagosomes form in developing myelin sheathes before trafficking from myelin to the OL soma. Mice with conditional OPC/OL-specific deletion of the essential autophagy gene Atg5 beginning on postnatal Day 5 develop a rapid tremor and die around postnatal Day 12. Further analysis revealed apoptotic death of OPCs, reduced differentiation, and reduced myelination. Surviving Atg5-/- OLs failed to produce proper myelin structure. In vitro, pharmacological inhibition of autophagy in OPC/dorsal root ganglion (DRG) co-cultures blocked myelination, producing OLs surrounded by many short processes. Conversely, autophagy stimulation enhanced myelination. These results implicate autophagy as a key regulator of OPC survival, maturation, and proper myelination. Autophagy may provide an attractive target to promote both OL survival and subsequent myelin repair after injury.

Subcutaneous Administration of PDGF-AA Improves the Functional Recovery After Spinal Cord Injury.

Previous studies by our group have demonstrated that the transplantation of exogenous platelet-derived growth factor (PDGF)-AA-overexpressing oligodendrocyte progenitor cells (OPCs) promotes tissue repair and recovery of neurological function in a rat model of spinal cord injury (SCI). However, it remains unclear whether treatment with PDGF-AA also affects endogenous oligodendrocytes (OLs) or even neurons, thus promoting further functional recovery after SCI. In the present study, we evaluated the therapeutic potential of PDGF-AA treatment by direct subcutaneous injection of PDGF-AA immediately after SCI. We demonstrated that PDGF-AA injection resulted in increased tissue sparing, myelination and functional recovery in rats following SCI. Further experimentation confirmed that PDGF-AA increased the survival of endogenous OPCs and OLs, and promoted the proliferation of OPCs and their differentiation into OLs. Moreover, PDGF-AA also protected motor neurons from death in the injured spinal cord. These results indicated that PDGF-AA administration may be an effective treatment for SCI.

Characterization of a murine mixed neuron-glia model and cellular responses to regulatory T cell-derived factors

One of the unmet clinical needs in demyelinating diseases such as Multiple Sclerosis (MS) is to provide therapies that actively enhance the process of myelin regeneration (remyelination) in the central nervous system (CNS). Oligodendrocytes, the myelinating cells of the CNS, play a central role in remyelination and originate from oligodendrocyte progenitor cells (OPCs). We recently showed that depletion of regulatory T cells (Treg) impairs remyelination in vivo, and that Treg-secreted factors directly enhance oligodendrocyte differentiation. Here we aim to further characterize the dynamics of Treg-enhanced oligodendrocyte differentiation as well as elucidate the cellular components of a murine mixed neuron-glia model.Murine mixed neuron-glia cultures were generated from P2–7 C57BL/6 mice and characterized for percentage of neuronal and glial cell populations prior to treatment at 7 days in vitro (div) as well as after treatment with Treg-conditioned media at multiple timepoints up to 12 div.Mixed neuron-glia cultures consisted of approximately 30% oligodendroglial lineage cells, 20% neurons and 10% microglia. Furthermore, a full layer of astrocytes, that could not be quantified, was present. Treatment with Treg-conditioned media enhanced the proportion of MBP+ oligodendrocytes and decreased the proportion of PDGFRα+ OPCs, but did not affect OPC proliferation or survival. Treg-enhanced oligodendrocyte differentiation was not caused by Treg polarizing factors, was dependent on the number of activation cycles Treg underwent and was robustly achieved by using 5% conditioned media.These studies provide in-depth characterization of a murine mixed neuron-glia model as well as further insights into the dynamics of Treg-enhanced oligodendrocyte differentiation.

Abstract TP103: Cholesterol Transporters Abca1 and Apoe Mediate Grey/White Matter Remodeling and Neurogenesis/Oligodendrogenesis After Stroke

The ATP-binding cassette transporter-A1 (ABCA1) and apolipoprotein-E (ApoE) are major cholesterol transporters in the brain. ABCA1 effluxes cholesterol and phospholipids to extra-cellular, and ApoE transport cholesterol and lipid back into cells. Brain-specific ABCA1-deficient (ABCA1 -B/-B ) mice exhibit reduced ApoE level in brain and cerebrospinal-fluid (CSF), and increased white matter damage and neurological deficits after stroke. Hypothesis: ApoE-treatment increases grey/white matter remodeling and neurogenesis/oligodendrogenesis in the ischemic brain in ABCA1 -B/-B -stroke mice. Methods: Adult ABCA1 -B/-B mice were subjected to distal middle cerebral artery occlusion (dMCAo) and were intraventricularly-infused with recombinant human ApoE2 (rhApoE2, 25μg in artificial-CSF) or artificial-CSF as vehicle-control by transplanting a micro-osmotic pump into the right lateral-ventricle starting 24h after dMCAo until sacrifice 14 days after dMCAo. Primary cultured cortical neurons (PCNs) and oligodendrocyte progenitor cells (OPCs) derived from ABCA1 -B/-B and ABCA1-floxed control (ABCA1 fl/fl ) mice were employed, and the PCN-axonal outgrowth and OPC proliferation/death were investigated. Results: No changes in the lesion volume between ABCA1 -B/-B +CSF and ABCA1 -B/-B +rhApoE2 mice were found. Compared with ABCA1 -B/-B +CSF mice, the ABCA1 -B/-B +rhApoE2 mice exhibit: 1) increased CSF ApoE level and improved functional outcome 7 and 14 days after dMCAo; 2) increased grey matter and white matter density in the ischemic boundary zone; 3) increased neurogenesis in the subventricular zone and oligodendrogenesis in the corpus collosum in the ischemic brain (p<0.05, n=11/group). In vitro data show that: 1) ABCA1 -B/-B decreased axonal-outgrowth and ApoE-treatment (2, 4 and 8 μg/ml) significantly attenuated ABCA1 -B/-B -induced reduction in axonal-outgrowth; 2) ABCA1 -B/-B decreased OPC proliferation and survival compared with ABCA1 fl/fl -OPCs and ApoE treatment increased OPC proliferation and OPC survival in primary cultured ABCA1 -B/-B -OPCs. Conclusion: ABCA1/ApoE contributes to grey and white matter remodeling, increases neurogenesis and oligodendrogenesis as well as improves functional recovery after stroke.

Co-Ultramicronized Palmitoylethanolamide/Luteolin Facilitates the Development of Differentiating and Undifferentiated Rat Oligodendrocyte Progenitor Cells.

Oligodendrocytes, the myelin-producing cells of the central nervous system (CNS), have limited capability to bring about repair in chronic CNS neuroinflammatory demyelinating disorders such as multiple sclerosis (MS). MS lesions are characterized by a compromised pool of undifferentiated oligodendrocyte progenitor cells (OPCs) unable to mature into myelin-producing oligodendrocytes. An attractive strategy may be to replace lost OLs and/or promote their maturation. N-palmitoylethanolamine (PEA) is an endogenous fatty acid amide signaling molecule with anti-inflammatory and neuroprotective actions. Recent studies show a co-ultramicronized composite of PEA and the flavonoid luteolin (co-ultraPEALut) to be more efficacious than PEA in improving outcome in CNS injury models. Here, we examined the effects of co-ultraPEALut on development of OPCs from newborn rat cortex cultured under conditions favoring either differentiation (Sato medium) or proliferation (fibroblast growth factor-2 and platelet-derived growth factor (PDGF)-AA-supplemented serum-free medium ("SFM")). OPCs in SFM displayed high expression of PDGF receptor alpha gene and the proliferation marker Ki-67. In Sato medium, in contrast, OPCs showed rapid decreases in PDGF receptor alpha and Ki-67 expression with a concomitant rise in myelin basic protein (MBP) expression. In these conditions, co-ultraPEALut (10μM) enhanced OPC morphological complexity and expression of MBP and the transcription factor TCF7l2. Surprisingly, co-ultraPEALut also up-regulated MBP mRNA expression in OPCs in SFM. MBP expression in all cases was sensitive to inhibition of mammalian target of rapamycin. Within the context of strategies to promote endogenous remyelination in MS which focus on enhancing long-term survival of OPCs and stimulating their differentiation into remyelinating oligodendrocytes, co-ultraPEALut may represent a novel pharmacological approach.

Co-transplantation of MRF-overexpressing oligodendrocyte precursor cells and Schwann cells promotes recovery in rat after spinal cord injury

Oligodendrocyte (OL) replacement is a promising treatment strategy for spinal cord injury (SCI). However, the poor survival of transplanted OLs or their precursors and inhibition of axonal regeneration are two major challenges with this approach. Our previous study showed that Schwann cells (SCs) promoted survival, proliferation, and migration of transplanted OL progenitor cells (OPCs) and neurological recovery. Remyelination is an important basis for functional recovery following spinal cord injury. It has been reported that myelin gene regulatory factor (MRF), a transcriptional regulator which specifically is expressed in postmitotic OLs within the CNS, is essential for OL maturation and CNS myelination. In the present study, we investigated whether co-transplantation of MRF-overexpressing OPCs (MRF-OPCs) and SCs could improve functional recovery in a rat model of contusional SCI. MRF overexpression had no effect on OPC survival or migration, but stimulated the differentiation of OPCs both in vitro and in vivo. Co-transplantation of MRF-OPCs and SCs increased myelination and tissue repair after SCI, leading to the recovery of neurological function. These results indicate that co-transplantation of MRF-OPCs and SCs may be an effective treatment strategy for SCI.

Abstract T P94: Differential Response to Treatment of Stroke in Rats With T1dm With Bone Marrow Stromal Cells Derived From Normal or T1DM Rats

Objective: Treatment of stroke with bone-marrow-stromal cells (BMSCs) derived from normal rats (Nor-BMSCs) at 24h after stroke onset improves functional recovery in non-DM rats, but not in type-one DM (T1DM) rats. In the present study, we tested the differential therapeutic effects and mechanisms of action of the treatment of T1DM stroke with BMSCs derived from DM rats (DM-BMSCs) or Nor-BMSCs. Methods: T1DM rats were subjected to 2h of middle cerebral artery occlusion (MCAo) and were treated with DM-BMSCs, Nor-BMSCs or vehicle control (n=8/group) initiated at 24h after MCAo. A battery of functional tests, and vascular and white matter (WM) changes and in vitro cell culture experiments were performed. Results: Compared with Nor-BMSCs, treatment of stroke in T1DM rats with DM-BMSCs significantly improved functional outcome. DM-BMSCs increased vascular remodeling identified by cerebral vascular density and cerebral artery density, increased Bielschowsky silver (axon marker) and Luxol fast blue (myelin marker) expression in the ischemic border zone (IBZ) compared to T1DM-MCAo rats (p<0.05). However, DM-BMSC treatment also increased artherosclerosis-like changes identified by an increased internal carotid wall thickness, artery intimae thickness, and decreased cerebral artery diameter (p<0.05) compared to T1DM-MCAo control. DM-BMSCs treatment significantly increased PDGF, but decreased RhoA and ROCK expression in the IBZ. In vitro, DM-BMSCs have higher miR133b (1.9 fold) expression compared to Nor-BMSCs. DM-BMSC conditioned medium increased brain endothelial cell, smooth muscle cell and oligodendrocyte progenitor cell survival, as well as increased subventricular zone explant cell migration and primary cortical neuron axon outgrowth compared to Nor-BMSC conditioned or control medium. Conclusion: Treatment of stroke in T1-DM rats with DM-BMSCs have dual effects on stroke, i.e. significantly increases neurovascular remodeling, improves neurological recovery, and induces artherosclerosis-like vascular changes. The miR133b/RhoA/ROCK pathway may play a role in the DM-BMSC induced neurorestorative effects.

Type 2 diabetes reduces the proliferation and survival of oligodendrocyte progenitor cells in ishchemic white matter lesions

Diabetes mellitus (DM) is a major risk factor for stroke and it exacerbates tissue damage after ischemic insult. Diabetes is one of the important causes of the progression of white matter lesion, however, the pathological mechanisms remain unclear. The present study evaluated the influences of type 2 DM on ischemic subcortical white matter injury and the recruitment of oligodendrocyte progenitor cells (OPCs) under chronic cerebral hypoperfusion using type 2 diabetic (db/db) mice. After bilateral common carotid artery stenosis (BCAS), the rarefaction in the white matter was more severe in db/db mice than in db/+ mice, and the number of glutathione S-transferase-pi (GST-pi)-positive mature oligodendrocytes (OLG) was lower in db/db mice than in db/+ mice at 4 and 8 weeks after ischemia. There were no significant differences in the number of single-stranded DNA (ssDNA)-positive apoptotic cells in the deep white matter between the db/db and db/+ mice. We found a transient increase in the platelet-derived growth factor receptor-α (PDGFRα)-positive OPCs in white matter lesions after ischemia. However, significantly fewer PDGFRα-positive OPCs were detected in db/db than db/+ mice from 4weeks after BCAS. The number of Ki67-positive proliferating cells in the deep white matter was significantly lower in db/db mice than in db/+ mice from 4 to 8weeks after BCAS. Most of the Ki67-positive cells were PDGFRα-positive OPCs. Finally, we assessed the survival of 5-bromo-2′-deoxyuridine (BrdU)-positive proliferating cells in ischemic white matter, and found significantly poorer survival of BrdU/PDGFRα-positive OPCs or BrdU/GST-pi-positive OLGs in the db/db mice compared to the db/+ mice in the white matter after BCAS. Our findings suggest that the type 2 DM mice exhibited more severe white matter injury 8 weeks after chronic ischemia. Decreased proliferation and survival of OPCs may play an important role in the progression of white matter lesions after ischemia in diabetics.

Oligodendrocyte Progenitor Cell Susceptibility to Injury in Multiple Sclerosis

Remyelination in multiple sclerosis (MS) is often incomplete. In experimental models, oligodendrocyte progenitor cells (OPCs) rather than previously myelinating oligodendrocytes (OLs) are responsible for remyelination. This study compares the relative susceptibility of adult human OPCs and mature OLs to injury in actively demyelinating MS lesions and under invitro stress conditions. In all lesions (n = 20), the number of OLs (Olig2 weak/NogoA positive) was reduced compared to control white matter (mean 38± 4% of control value). In 11 cases, OPC numbers (Olig2 strong; NogoA negative) were also decreased; in eight of these, the reduction was greater for OPCs than for OLs. In the other nine samples, OPC numbers were greater than control white matter, indicating ongoing OPC migration and/or proliferation. Analysis of co-cultures with rat dorsal root ganglia neurons confirmed that OPCs were more capable of contacting and ensheathing axons than OLs. In isolated culture under stress conditions (withdrawal of serum/glucose and/or antioxidants), OPCs showed increased cell death and reduced process extension compared to OLs. Under all culture conditions, OPCs up-regulated expression of genes in the extrinsic proapoptotic pathway, and had increased susceptibility to tumor necrosis factor-induced cell death as compared to OLs. Our data suggest that susceptibility of OPCs to injury within the MS lesion environment contributes to the limited remyelination in MS.

Investigation of Sequential Growth Factor Delivery during Cuprizone Challenge in Mice Aimed to Enhance Oligodendrogliogenesis and Myelin Repair

Repair in multiple sclerosis involves remyelination, a process in which axons are provided with a new myelin sheath by new oligodendrocytes. Bone morphogenic proteins (BMPs) are a family of growth factors that have been shown to influence the response of oligodendrocyte progenitor cells (OPCs) in vivo during demyelination and remyelination in the adult brain. We have previously shown that BMP4 infusion increases numbers of OPCs during cuprizone-induced demyelination, while infusion of Noggin, an endogenenous antagonist of BMP4 increases numbers of mature oligodendrocytes and remyelinated axons following recovery. Additional studies have shown that insulin-like growth factor-1 (IGF-1) promotes the survival of OPCs during cuprizone-induced demyelination. Based on these data, we investigated whether myelin repair could be further enhanced by sequential infusion of these agents firstly, BMP4 to increase OPC numbers, followed by either Noggin or IGF-1 to increase the differentiation and survival of the newly generated OPCs. We identified that sequential delivery of BMP4 and IGF-1 during cuprizone challenge increased the number of mature oligodendrocytes and decreased astrocyte numbers following recovery compared with vehicle infused mice, but did not alter remyelination. However, sequential delivery of BMP4 and Noggin during cuprizone challenge did not alter numbers of oligodendrocytes or astrocytes in the corpus callosum compared with vehicle infused mice. Furthermore, electron microscopy analysis revealed no change in average myelin thickness in the corpus callosum between vehicle infused and BMP4-Noggin infused mice. Our results suggest that while single delivery of Noggin or IGF-1 increased the production of mature oligodendrocytes in vivo in the context of demyelination, only Noggin infusion promoted remyelination. Thus, sequential delivery of BMP4 and Noggin or IGF-1 does not further enhance myelin repair above what occurs with delivery of Noggin alone.