Myelination Trajectory and Microglial Dynamics Following Repeated Sevoflurane Exposure in Developing Brain.

Excessive release of glutamate, oxidative stress, inflammation after ischemic brain injury can lead to demyelination. Astrocytes participate in the maturation and differentiation of oligodendrocyte precursor cells (OPCs), and play multiple roles in the process of demyelination and remyelination. Here, we studied the role of Astrocyte-derived exosomes (AS-Exo) under ischemic conditions in proliferation, differentiation and migration of OPCs in vitro. Exosomes were collected from astrocytes supernatant by differential centrifugation from control astrocytes (CTexo), mild hypoxia astrocytes (O2R24exo) which were applied oxygen-glucose deprivation for 2h and reperfusion for 24h (OGD2hR24h) and severe hypoxia astrocytes (O4R24exo) which were applied oxygen-glucose deprivation for 4h and reperfusion for 24h (OGD4hR24h). Exosomes (20µg/ml) were co-cultured with OPCs for 24h and their proliferation, differentiation and migration were detected. The results showed that AS-Exo under severe hypoxia (O4R24exo) inhibit the proliferation of OPCs. Meanwhile, all exosomes from three groups can promote OPCs differentiation and migration. Compared to control, the expressions of MAG and MBP, markers of mature oligodendrocytes, were significantly increased in AS-Exo treatment groups. AS-Exo treatment significantly increased chemotaxis for OPCs. AS-Exo improve OPCs' differentiation and migration, whereas AS-Exo with severe hypoxic precondition suppress OPCs' proliferation. AS-Exo may be a potential therapeutic target for myelin regeneration and repair in white matter injury or other demyelination related diseases.

The massive loss of oligodendrocytes caused by various pathological factors is a basic feature of many demyelinating diseases of the central nervous system (CNS). Based on a variety of studies, it is now well established that impairment of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is a vital event in the failed treatment of demyelinating diseases. Recent evidence suggests that Foxg1 is essential for the proliferation of certain precursors and inhibits premature neurogenesis during brain development. To date, very little attention has been paid to the role of Foxg1 in the proliferation and differentiation of OPCs in demyelinating diseases of the CNS. Here, for the first time, we examined the effects of Foxg1 on demyelination and remyelination in the brain using a cuprizone (CPZ)-induced mouse model. In this work, 7-week-old Foxg1 conditional knockout and wild-type (WT) mice were fed a diet containing 0.2% CPZ w/w for 5 weeks, after which CPZ was withdrawn to enable remyelination. Our results demonstrated that, compared with WT mice, Foxg1-knockout mice exhibited not only alleviated demyelination but also accelerated remyelination of the demyelinated corpus callosum. Furthermore, we found that Foxg1 knockout decreased the proliferation of OPCs and accelerated their differentiation into mature oligodendrocytes both in vivo and in vitro. Wnt signaling plays a critical role in development and in a variety of diseases. GSK-3β, a key regulatory kinase in the Wnt pathway, regulates the ability of β-catenin to enter nuclei, where it activates the expression of Wnt target genes. We then used SB216763, a selective inhibitor of GSK-3β activity, to further demonstrate the regulatory mechanism by which Foxg1 affects OPCs in vitro. The results showed that SB216763 clearly inhibited the expression of GSK-3β, which abolished the effect of the proliferation and differentiation of OPCs caused by the knockdown of Foxg1. These results suggest that Foxg1 is involved in the proliferation and differentiation of OPCs through the Wnt signaling pathway. The present experimental results are some of the first to suggest that Foxg1 is a new therapeutic target for the treatment of demyelinating diseases of the CNS.

Because successful remyelination does not occur following traumatic spinal cord injury, patients suffer from long tract dysfunction. However, demyelination is followed by remyelination in early multiple sclerosis. Oligodendrocyte precursor cells constitute a large cell population in the adult mammalian central nervous system. We demonstrated the proliferation, migration, and differentiation of oligodendrocyte precursor cells in chemically induced demyelination, a model for multiple sclerosis, and reported that Nkx2.2 expression may regulate oligodendrocyte precursor cell differentiation, making it a key factor in the differentiation. To investigate what factors disturb remyelination in spinal cord injury, we examined the oligodendrocyte precursor cell proliferation and differentiation, and the expression of Nkx2.2 using contusive injury in rats as a model for traumatic spinal cord injury. This study showed that oligodendrocyte precursor cells proliferated after contusive injury but did not subsequently differentiate. The number of Nkx2.2-positive oligodendrocyte precursor cells did not significantly change in the tissue surrounding the lesion. Within the demyelinating lesion, the peak of Nkx2.2-positive oligodendrocyte precursor cell was delayed, and its level was lower than in the chemical models. No clearly recognizable oligodendrocytes were found in the demyelinating lesion throughout the observation period. To assess whether environmental changes differ between these two models, mRNA expressions of various cytokines were evaluated and compared. IL-1beta and IL-6 mRNA significantly increased in the contusion-induced injury model, 6 h after the injury. These results suggest that environmental factors such as cytokines may affect Nkx2.2 expression or oligodendrocyte precursor cell differentiation in the contusion-induced spinal cord injury model.

Effects of hypothermia on oligodendrocyte precursor cell proliferation, differentiation and maturation following hypoxia ischemia in vivo and in vitro

MAZ regulates ferroptosis, apoptosis and differentiation of oligodendrocyte precursor cells

Differentiation of rat oligodendrocyte precursor cells in chemical conditional medium in vitro

Post-stroke functional recovery can occur spontaneously during the subacute phase; however, how post-stroke fibrotic repair affects functional recovery is highly debated. Platelet-derived growth factor receptor β (PDGFRβ)-expressing pericytes are responsible for post-stroke fibrotic repair within infarct areas; therefore, we examined peri-infarct neural reorganization and functional recovery after permanent middle cerebral artery occlusion (pMCAO) using pericyte-deficient Pdgfrb+/- mice. Time-dependent reduction of infarct area sizes, i.e., repair, was significantly impaired in Pdgfrb+/- mice with recovery of cerebral blood flow (CBF) in ischemic areas attenuated by defective leptomeningeal arteriogenesis and intrainfarct angiogenesis. Peri-infarct astrogliosis, accompanied by increased STAT3 phosphorylation, was attenuated in Pdgfrb+/- mice. Pericyte-conditioned medium (PCM), particularly when treated with platelet-derived growth factor subunit B (PDGFB) homodimer (PDGF-BB; PCM/PDGF-BB), activated STAT3 and enhanced the proliferation and activity of cultured astrocytes. Although peri-infarct proliferation of oligodendrocyte (OL) precursor cells (OPCs) was induced promptly after pMCAO regardless of intrainfarct repair, OPC differentiation and remyelination were significantly attenuated in Pdgfrb+/- mice. Consistently, astrocyte-CM (ACM) promoted OPC differentiation and myelination, which were enhanced remarkably by adding PCM/PDGF-BB to the medium. Post-stroke functional recovery correlated well with the extent and process of intrainfarct repair and peri-infarct oligodendrogenesis. Overall, pericyte-mediated intrainfarct fibrotic repair through PDGFRβ may promote functional recovery through enhancement of peri-infarct oligodendrogenesis as well as astrogliosis after acute ischemic stroke.

Perinatal white matter injury (WMI), which is prevalent in premature infants, involves M2 microglia affecting oligodendrocyte precursor cells (OPCs) through exosomes, promoting OPC growth and reducing WMI. The molecular mechanism of WMI remains unclear, and this study explored the role of M2 microglia-derived exosomes in WMI. A tMCAO rat model was constructed to simulate WMI characteristics in vivo. Cresyl violet staining, neurobehavioral tests, rotarod tests, immunofluorescence and immunochemistry were used to assess the role of exos-derived miR-144-5p in pathological and neurological changes in rats. OGD/R cellular models were constructed to mimic WMI characteristics in vitro. CCK-8, TUNEL, Western blotting and immunofluorescence were used to assess the role of exos-derived miR-144-5p in OPC phenotypes. Rescue assays were used to assess the role of the PTEN/AKT pathway in miR-144-5p-mediated OPC phenotypes. Bioinformatics and mechanistic experiments were used to assess the association of PTEN or KLF12 with miR-144-5p in OPCs. M2-Exos suppressed cerebral injury and facilitated demyelination repair in rats post WMI. M2-Exos suppressed OGD/R-stimulated OPC apoptosis and facilitated OGD/R-stimulated OPC differentiation. M2-Exo-derived miR-144-5p suppressed OGD/R-stimulated OPC apoptosis and facilitated OGD/R-stimulated OPC differentiation. M2-Exo-derived miR-144-5p suppressed cerebral injury and facilitated demyelination repair in rats post WMI. MiR-144-5p suppressed OGD/R-stimulated OPC apoptosis and facilitated OGD/R-stimulated OPC differentiation through PTEN downregulation. MiR-144-5p targeted the KLF12 3'UTR to repress PTEN transcription in OPCs. M2 microglia secrete miR-144-5p to reduce WMI by targeting KLF12 in OPCs, inhibiting PTEN/AKT pathway activity, and offering potential targeted therapeutic insights for WMI.

Biomaterial drug delivery systems (DDS) can be used to regulate growth factor release and combat the limited intrinsic regeneration capabilities of central nervous system (CNS) tissue following injury and disease. Of particular interest are systems that aid in oligodendrocyte regeneration, as oligodendrocytes generate myelin which surrounds neuronal axons and helps transmit signals throughout the CNS. Oligodendrocyte precursor cells (OPCs) are found in small numbers in the adult CNS, but are unable to effectively differentiate following CNS injury. Delivery of signaling molecules can initiate a favorable OPC response, such as proliferation or differentiation. Here, we investigate the delivery of one such molecule, platelet derived growth factor-AA (PDGF-AA), from poly(lactic-co-glycolic) acid microparticles to OPCs in a 3D polyethylene glycol-based hydrogel. The goal of this DDS was to better understand the relationship between PDGF-AA release kinetics and OPC fate. The system approximates native brain tissue stiffness, while incorporating PDGF-AA under seven different delivery scenarios. Within this DDS, supply of PDGF-AA followed by PDGF-AA withdrawal caused OPCs to upregulate gene expression of myelin basic protein (MBP) by factors of 1.6-9.2, whereas continuous supply of PDGF-AA caused OPCs to remain proliferative. At the protein expression level, we observed an upregulation in O1, a marker for mature oligodendrocytes. Together, these results show that burst release followed by withdrawal of PDGF-AA from a hydrogel DDS stimulates survival, proliferation, and differentiation of OPCs in vitro. Our results could inform the development of improved neural regeneration strategies that incorporate delivery of PDGF-AA to the injured CNS. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2402-2411, 2018.

Although transgenic and knockout mice are widely used to study the specification and differentiation of oligodendrocyte precursor cells (OPCs), mouse primary OPCs are difficult to be purified and maintained, and many in vitro studies have to resort to rat OPCs as substitutes. In this study, we reported that mouse O4 negative early-stage OPCs can be obtained by culturing cortical tissue blocks, and the simultaneous treatment of OPCs with Platelet Derived Growth Factor-AA (PDGFaa), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) is the key for the propagation of mouse OPCs in culture. EGF was found to be a potent mitogen for OPCs and cooperate with PDGFaa to extend cell division and inhibit their differentiation. EGF also collaborates with PDGFaa and bFGF to convert bipolar or tripolar OPCs to more vital fibroblast-like OPCs without compromising their oligodendrocyte differentiation potential. In addition, EGF promoted the survival and proliferation of glial progenitor cells (GPCs) derived from primary OPC cultures, and a mixture of GPCs and OPCs can be obtained and propagated in the presence of EGF, bFGF, and PDGFaa. Once EGF is withdrawn, GPC population decreased sharply and fibroblast-like OPCs changed into typical OPCs morphology, then homogeneous OPCs were obtained subsequently.

Intermittent theta-burst stimulation alleviates hypoxia-ischemia-caused myelin damage and neurologic disability

The aim of the present study has been to obtain high yields of oligodendrocyte precursor cells (OPCs) in culture. This is a first step in facilitation of myelin repair. We show that, in addition to factors, known to promote proliferation, such as basic fibroblast growth factor (FGF-2) and platelet derived growth factor (PDGF) the choice of the basal medium exerts a significant influence on the yield of OPCs in cultures from newborn rats. During a culture period of up to 9 days we observed larger numbers of surviving cells in Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute Medium (RPMI) compared with Neurobasal Medium (NB). A larger number of A2B5-positive OPCs was found after 6 days in RPMI based media compared with NB. The percentage of bromodeoxyuridine (BrdU)-positive cells was largest in cultures maintained in DMEM and RPMI. The percentage of caspase-3 positive cells was largest in NB, suggesting that this medium inhibits OPC proliferation and favors apoptosis. A difference between NB and DMEM as well as RPMI is the reduced Na+-content. The addition of equiosmolar supplements of mannitol or NaCl to NB medium rescued the BrdU-incorporation rate. This suggested that the osmolarity influences the proliferation of OPCs. Plating density as well as residual microglia influence OPC survival, BrdU incorporation, and caspase-3 expression. We found, that high density cultures secrete factors that inhibit BrdU incorporation whereas the presence of additional microglia induces an increase in caspase-3 positive cells, indicative of enhanced apoptosis. An enhanced number of microglia could thus also explain the stronger inhibition of OPC differentiation observed in high density cultures in response to treatment with the cytokines TNF-α and IFN-γ. We conclude that a maximal yield of OPCs is obtained in a medium of an osmolarity higher than 280 mOsm plated at a relatively low density in the presence of as little microglia as technically achievable.

Low frequency pulsed electromagnetic field promotes differentiation of oligodendrocyte precursor cells through upregulation of miR-219-5p in vitro

One of the most frequent psychological consequences of stroke is depression. Previous animal studies have demonstrated that post-conditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury. Thus, we hypothesized that repeated exposure to sevoflurane after transient ischemia can prevent the development of depressive-like behavior. To test this hypothesis, we induced transient cerebral ischemia via transient occlusion of bilateral common carotid arteries and examined the effects of subsequent repeated exposure to sevoflurane on sucrose preference, locomotor activity, and rearing activity in rats. To explore the putative neurobiological mechanisms, we further investigated the roles of hippocampal CB1 receptor in the behavioral effects of sevoflurane. We found that repeated sevoflurane exposures reversed ischemia-induced depressive-like behaviors. Furthermore, CB1 receptor inhibition in the dorsal hippocampus (DH) abolished the effects of sevoflurane exposures on ischemia-induced depressive-like behaviors. Inaddition, repeated sevoflurane exposures increased CB1 receptor expression and endocannabinoids levels in the DH of ischemic rats. Moreover, repeated sevoflurane exposures enhanced the expression of β-arrestin 2, increased the activation of extracellular signal-regulated kinases (ERK)1/2, and promoted the interaction of β-arrestin 2 and ERK1/2 in theDH, and such effects were reversed by CB1 receptor antagonism in the DH. Finally, β-arrestin 2 expression and ERK1/2 activation in the DH were critical for the preventativeeffects of sevoflurane exposures on ischemia-induced depressive-like behaviors. Taken together, our results suggested that sevoflurane exposure after brain ischemia may prevent the development of depression, and such preventativeeffects of sevoflurane are likely ascribed to the activation ofCB1 receptor-mediated β-arrestin 2-ERK1/2 signaling pathways. We propose that the following mechanisms are critical for the preventative effects of sevoflurane against post-stroke depressive and anxiety behaviors: repeated sevoflurane exposure after transient brain ischemia enhances N-arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) levels and normalize cannabinoid receptor type 1 (CB1) receptor expression in the dorsal hippocampus, which results in enhanced interaction of β-arrestin 2 and extracellular signal-regulated kinases (ERK1/2) and increased ERK1/2 activation, leading to decreased depressive and anxiety behaviors. We think these findings should provide a new strategy for treatment of post-stroke depression.

Background and Purpose- Type 2 diabetes mellitus (T2DM) is a major comorbidity that exacerbates ischemic brain injury and worsens functional outcome after stroke. T2DM is known to aggravate white matter (WM) impairment, but the underlying mechanism is not completely understood. This study was designed to test the hypothesis that T2DM impedes poststroke WM recovery by suppressing both oligodendrogenesis and beneficial microglia/macrophage responses. Methods- Permanent distal middle cerebral artery occlusion was performed in wild-type, homozygous diabetic db/db, and heterozygous db/+ mice. The adhesive removal, open field, and Morris water maze tests were used to assess neurobehavioral outcomes. Neuronal tissue loss, WM damage, oligodendrogenesis, and microglia/macrophage responses were evaluated up to 35 days after stroke. The functional integrity of WM was measured by electrophysiology. Primary microglia-oligodendrocyte cocultures were used for additional mechanistic studies. Results- T2DM exacerbated structural damage and impaired conduction of compound action potentials in WM 35 days after stroke. The deterioration in WM integrity correlated with poor sensorimotor performance. Furthermore, T2DM impaired the proliferation of oligodendrocyte precursor cells and the generation of new myelinating oligodendrocytes. T2DM also promoted a shift of microglia/macrophage phenotype toward the proinflammatory modality. Coculture studies confirmed that microglia/macrophage polarization toward the proinflammatory phenotype under high glucose conditions suppressed oligodendrocyte precursor cell differentiation. Conclusions- Deterioration of WM integrity and impairments in oligodendrogenesis after stroke are associated with poor long-term functional outcomes in experimental diabetes mellitus. High glucose concentrations may shift microglia/macrophage polarization toward a proinflammatory phenotype, significantly impairing oligodendrocyte precursor cell differentiation and WM repair.