MiR-146a promotes remyelination in a cuprizone model of demyelinating injury.

The death of mature oligodendrocytes (OLs) which are the sole myelinating cells of the central nervous system (CNS), leads to demyelination and functional deficits. Currently, there is lack of effective remyelination therapies for patients with demyelinating diseases. MicroRNAs (miRNAs) mediate OL function. We hypothesized that miR-146a, by inactivating interleukin-1 receptor-associated kinase 1 (IRAK1), promotes differentiation of oligodendrocyte progenitor cells (OPCs) and thereby enhances remyelination. To test this hypothesis, a demyelination model induced by a cuprizone (CPZ) diet was employed, in which C57BL/6J mice were fed with a CPZ diet for 5weeks. After termination of CPZ diet, the mice were randomly treated with continuous infusion of miR-146a mimics or mimic controls into the corpus callosum for 7days. Compared to the mimic control, infusion of miR-146a mimics facilitated remyelination assessed by increased myelin basic proteins in the corpus callosum, which was associated with augmentation of newly generated mature OLs. Infusion of miR-146a mimics also substantially elevated miR-146a levels in the corpus callosum and fluorescently tagged miR-146a mimics were mainly detected in OPCs. Western blot and double immmunofluorescent staining analysis showed that the miR-146a treatment considerably reduced IRAK1 protein levels and the number of IRAK1-positive cells, respectively. Collectively, these data indicate that exogenous miR-146a enhances remyelination, possibly by promoting OPCs to differentiate into myelinated OLs via targeting IRAK1.

Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). No effective remyelination therapies are in use. We hypothesized that thymosin beta4 (Tß4) is an effective remyelination treatment by promoting differentiation of oligodendrocyte progenitor cells (OPCs), and that the epidermal growth factor receptor (EGFR) signaling pathway contributes to this process. Two demyelination animal models were employed in this study: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated daily for 30days, with Tß4 or saline treatment initiated on the day of EAE onset; and 2) cuprizone diet model, a non-inflammatory demyelination model. The mice were treated daily for 4weeks with Tß4 or saline after fed a cuprizone diet for 5weeks. Immunofluorescent staining and Western blot were performed to measure the differentiation of OPCs, myelin and axons, respectively. To obtain insight into mechanisms of action, the expression and activation of the EGFR pathway was measured. AG1478, an EGFR inhibitor, was employed in a loss-of-function study. Data revealed that animals in both demyelination models exhibited significant reduction of myelin basic protein (MBP(+)) levels and CNPase(+) oligodendrocytes. Treatment of EAE mice with Tß4 significantly improved neurological outcome. Double immunofluorescent staining showed that Tß4 significantly increased the number of newly generated oligodendrocytes identified by BrdU(+)/CNPase(+) cells and MBP(+) mature oligodendrocytes, and reduced axonal damage in the EAE mice compared with the saline treatment. The newly generated mature oligodendrocytes remyelinated axons, and the increased mature oligodendrocytes significantly correlated with functional improvement (r=0.73, p<0.05). Western blot analysis revealed that Tß4 treatment increased expression and activation of the EGFR pathway. In the cuprizone demyelination model, Tß4 treatment was confirmed that significantly increased OPC differentiation and remyelination, and increased the expression of EGFR and activated the EGFR pathway in the demyelinating corpus callosum. In cultured OPCs, blockage of the activation of the EGFR pathway with AG1478 abolished the Tß4-increased OPC differentiation. Collectively, these findings indicate that: 1) Tß4 increases proliferation of OPCs and the maturation of OPCs to myelinating oligodendrocytes which in concert, likely contribute to the beneficial effect of Tß4 on EAE, 2) EGFR upregulated and activated by Tß4 may mediate the process of OPC differentiation, and 3) Tß4 could potentially be developed as a therapy for MS patients, and for other demyelinating neurological disorders.

Fingolimod treatment promotes proliferation and differentiation of oligodendrocyte progenitor cells in mice with experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a major demyelinating disease of the central nervous system (CNS) leading to functional deficits. The remyelination process is mediated by oligodendrocyte progenitor cells (OPCs). In this study, we tested the hypothesis that Fingolimod, a sphingosine 1-phosphate (S1P) receptor modulator, stimulates OPC differentiation into mature oligodendrocytes, in addition to its well-known anti-inflammatory effect. Using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we performed a dose-response study of Fingolimod (0.15 or 0.3mg/kg bw), which was initiated on the day of EAE onset. The neurological function was tested to determine the optimal dose of Fingolimod. Immunofluorescent staining was performed to measure the profile of OPC proliferation and differentiation. The mechanistic premise underlying the therapeutic effect of Fingolimod, was that Fingolimod stimulates the sonic hedgehog (Shh) pathway, and this pathway promotes OPC differentiation. To test this hypothesis, a loss-of-function study using cyclopamine, an inhibitor of the sonic hedgehog (Shh) pathway, was employed in vivo. Protein levels of the Shh pathway were measured by Western blot analysis. We found that Fingolimod treatment (0.3mg/kg bw) significantly decreased cumulative disease score compared to the EAE control group. Concurrently, OPCs and proliferation of OPCs were significantly increased in the white matter of the brain and spinal cord at day 7 and day 30 after EAE onset, and oligodendrocytes, myelination and differentiation of OPCs were significantly increased at day 30 compared with the EAE control group. EAE mice treated with Fingolimod exhibited substantially elevated levels of Shh, its receptor Smoothened and effector Gli1 in the white matter of the CNS. However, combination treatment of EAE mice with cyclopamine-Fingolimod decreased Fingolimod monotherapy elevated protein levels of Smoothened and Gli1, and abolished the effect of Fingolimod on OPC proliferation and differentiation, as well as on neurological function outcome. Together, these data demonstrate that Fingolimod is effective as a treatment of EAE by promoting OPC proliferation and differentiation, which facilitate remyelination. In addition, the Shh pathway likely contributes to the therapeutic effects of Fingolimod on OPCs.

Perfusion and Diffusion Abnormalities of Multiple Sclerosis Lesions and Relevance of Classified Lesions to Disease Status.

OBJECTIVE: Hemodynamic abnormality and disruption of white matter (WM) integrity are significant components in the pathophysiology of multiple sclerosis (MS) lesions. However, the roles of stratified lesions with distinct degrees of hemodynamic and structural injury in disease states remain to be explored. We tested the hypothesis that hemodynamic and structural impairment, as assessed by cerebral blood volume (CBV) and fractional anisotropy (FA), respectively, characterizes the extent of tissue injury, and the load of lesion with substantial tissue destruction would reflect the disease status and therefore, would be related to clinical disability. METHODS: Seven relapsing-remitting MS patients and seven healthy controls underwent perfusion, diffusion and conventional MRI scans. Based on T2-FLAIR and T1-weighted image, WM plaques were classified. After image coregistration, values of CBV and FA were estimated in three distinct lesion types (active, T1-hypointense and T1-isointense lesion) and compared with those obtained in WM from controls. A total of 1135 lesions were evaluated. Brain volumetric measurement and correlative analysis between brain atrophy, lesion volume and clinical disability were also performed. RESULTS: Compared with normal WM, significantly reduced CBV and FA were present in the T1-hypointense lesion, while insignificant changes in both parameters were exhibited in the T1-isointense lesion. However, increased CBV but significantly decreased FA was detected in the active lesion. A close spatial relationship between active and T1-hypointense lesion was observed. Lesion load represented by T1-hypointense plus active lesion volume significantly correlated with brain atrophy, which, in turn, significantly correlated with the severity of clinical disability. CONCLUSION: A distinct combination of CBV and FA characterizes the status of a specific lesion type. A severe structural impairment does not solely occur in the T1-hypointense lesion, but is also associated with the active lesion. The burden of the lesion with extensive structural damage provides an image index, indicative of disease status.

The appointment of neurointensivists is financially beneficial to the employer.

BACKGROUND: Although the impact of a neurointensivist (NI) on patient outcomes has been examined in the past, the financial impact has not been estimated before. METHODS: We extracted the financial data from the Neuro-Intensive Care Unit (NICU) at Henry Ford Hospital during two 3-year periods, one before and one after the appointment of a NI. Net revenue (NR), total direct expenses (TDE), and contribution margin (CM) were compared between these two periods both for Henry Ford Hospital and the Henry Ford Medical Group. RESULTS: The average number of admissions increased by 24% during the period when the NI was present, the number of patient-days by 25% and the average length of stay by 2%. In the second period, when the NI was billing for critical care time spent in the NICU, as well as for procedures he performed, the mean yearly NR was $402,000, the TDE $317,000 and the NR/TDE 1.24 (>1.0 represents profitability). The combined mean NR (Henry Ford Hospital + Medical Group) increased by 54.6%, the combined TDE by 42.2% and the combined CM by 91.2% in the period when the NI was present. This is reflected in the combined mean CM per admission, which also increased by 56.4% in the after period. CONCLUSION: This study shows a significant financial benefit for the Henry Ford Health System during the period when a NI was present in the NICU.

Bone marrow stromal cells increase oligodendrogenesis after stroke.

Oligodendrocytes are sensitive to ischemic damage. The Sonic hedgehog (Shh) pathway is critical in oligodendrogenesis; Gli1 is the principal effector of Shh signaling. We investigated oligodendrogenesis and Shh/Gli1 pathway activation after bone marrow stromal cell (BMSC) treatment of stroke in rats. Rats were subjected to the middle cerebral artery occlusion (MCAo). BMSCs have been shown to promote functional recovery post stroke. A therapeutic dose of BMSC (3 x 10(6) cells) treatment was initiated 1 day after MCAo. Immunohistochemistry was carried out to measure the oligodendrocyte progenitor cells, oligodendrocytes, myelin, and expressions of Shh and Gli1 at 14 days after MCAo. Gene expression of Shh and Gli1 was tested at 2 days after MCAo. An in vitro study was used to investigate the effects of BMSC on a premature oligodendrocyte cell line (N20.1 cells). BMSC treatment significantly increased O4(+) oligodendrocytes, MBP(+) area, and bromodeoxyuridine (BrdU)(+), NG2(+), BrdU(+)-NG2(+) cells, and mRNA and protein expressions of Shh and Gli1 in the ipsilateral brain of the MCAo rats than that in phosphate buffered saline (PBS)-treated rats. BMSCs promoted N20.1 cell proliferation and Gli1 mRNA expression, and these effects were abolished by the Shh pathway inhibitor cyclopamine. These data indicate that the BMSC treatment stimulates oligodendrogenesis by activation of the Shh/Gli1 pathway post stroke.

Bone marrow stromal cell therapy reduces proNGF and p75 expression in mice with experimental autoimmune encephalomyelitis.

Demyelination is prominent in experimental autoimmune encephalomyelitis (EAE). The receptor p75 and its high affinity ligand proNGF are required for oligodendrocyte death after injury. We hypothesize that bone marrow stromal cells (BMSCs) provide therapeutic benefit in EAE mice by reducing proNGF/p75 expression. PBS or BMSCs (2 x 10(circumflex)6) were administered intravenously on the day of EAE onset. Neurological function and demyelination areas were measured. Immunohistochemical staining was used to measure apoptotic oligodendrocytes, expression of proNGF and p75, and the relationship between proNGF and p75 in neural cells. proNGF was used to treat oligodendrocytes in culture with or without BMSCs. EAE mice exhibited neurological function deficit and demyelination, and expression of proNGF and p75 was increased. BMSC treatment improved functional recovery, reduced demyelination area and apoptotic oligodendrocytes, decreased expression of proNGF and p75 compared with PBS treatment. proNGF(+) cells colocalized with neural cell markers, while p75 colocalized with an oligodendrocytic marker, and proNGF colocalized with p75. proNGF induced apoptosis of oligodendrocytes in vitro, and p75 antibody blocked this apoptotic activity. BMSCs reduced p75 expression and apoptotic activity in oligodendrocytes with proNGF treatment. BMSC treatment benefits on EAE mice may be fostered by decreasing the cellular expression of proNGF and p75, thereby reducing oligodendrocyte death.

Niaspan treatment improves neurological functional recovery in experimental autoimmune encephalomyelitis mice.

We investigated the treatment of experimental autoimmune encephalomyelitis (EAE) in mice with Niaspan, an agent used to elevate high-density lipoprotein (HDL). EAE mice were treated with Niaspan starting on the immunization or clinical onset day. Neurological functional recovery was significantly increased in the Niaspan treated mice (100 mg/kgbw) compared to the controls. Inflammatory infiltrates were significantly reduced in the Niaspan treatment group compared to the EAE controls. HDL level, intact myelin area, newly formed oligodendrocytes, regenerating axons, gene and protein levels of sonic hedgehog (Shh)/Gli1 were significantly increased in the Niaspan treated mice compared to EAE controls. These data indicate that Niaspan treatment improved functional recovery after EAE, possibly, via reducing inflammatory infiltrates and demyelination areas, and stimulating oligodendrogenesis and axonal regeneration. Niaspan-mediated activation of Shh/Gli1 pathway may promote functional recovery post-EAE.

Bone marrow stromal cells protect oligodendrocytes from oxygen-glucose deprivation injury.

Oligodendrocyte (OLG) damage leads to demyelination, which is frequently observed in ischemic cerebrovascular diseases. In this study, we investigated the effect of bone marrow stromal cells (BMSCs) on OLGs subjected to oxygen-glucose deprivation (OGD). N20.1 cells (mouse OLG cell line) were transferred into an anaerobic chamber for 3 hr in glucose-free and serum-free medium. After OGD incubation, OLG cultures were divided into the following groups: 1) OGD alone, 2) OLG cocultured with BMSCs, 3) treatment with the phosphoinostide 3-kinase (PI3k) inhibitor LY294002, 4) LY294002-treated OLGs with BMSC cocultured, and 5) anti-p75 antibody-treated OLGs. After an additional 3 hr of reoxygenation incubation, OLG viability and apoptosis were measured. The mRNA expression in the BMSCs and OLGs was analyzed using quantitative real-time PCR (RT-PCR). Serine/threonine-specific protein kinase (Akt), phosphorylated Akt (p-Akt), p75, and caspase 3 protein expressions in OLGs were measured by Western blot. Our results suggest that BMSCs produce growth factors, activate the Akt pathway, and increase the survival of OLGs. BMSCs also reduce p75 and caspase 3 expressions in the OGD-OLGs, which leads to decreased OLG apoptosis. BMSCs participate in OLG protection that may occur with promoting growth factors/PI3K/Akt and inhibiting the p75/caspase pathways. Our study provides insight into white matter damage and the therapeutic benefits of BMSC-based remyelinating therapy after stroke and demyelinating diseases.

Bone marrow stromal cells reduce axonal loss in experimental autoimmune encephalomyelitis mice.

We investigated the ability of human bone marrow stromal cell (hBMSC) treatment to reduce axonal loss in experimental autoimmune encephalomyelitis (EAE) mice. EAE was induced in SJL/J mice by injection with proteolipid protein (PLP). Mice were injected intravenously with hBMSCs or PBS on the day of clinical onset, and neurological function was measured daily (score 0-5) until 45 weeks after onset. Mice were sacrificed at week 1, 10, 20, 34, and 45 after clinical onset. Bielshowsky silver was used to identify axons. Immunohistochemistry was performed to measure the expression of nerve growth factor (NGF) and MAB1281, a marker of hBMSCs. hBMSC treatment significantly reduced the mortality, the disease severity, and the number of relapses in EAE mice compared with PBS treatment. Axonal density and NGF(+) cells in the EAE brain were significantly increased in the hBMSC group compared with the PBS group at 1, 10, 20, 34, and 45 weeks. Disease severity was significantly correlated with decreased axonal density and decreased NGF, and increased axonal density was significantly correlated with reduced loss of NGF expression after hBMSC treatment. Most of the NGF(+) cells are brain parenchymal cells. Under 5% of MAB1281(+) cells colocalized with NG2(+), a marker of oligodendrocyte progenitor cells. Nearly 10% of MAB1281(+) cells colocalized with GFAP, a marker of astrocytes, and MAP-2, a marker of neurons. Our findings indicate that hBMSCs improve functional recovery and may provide a potential therapy aimed at axonal protection in EAE mice, in which NGF may play a vital role.

Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice.

We investigated the treatment of remitting-relapsing experimental autoimmune encephalomyelitis (EAE) in mice with human bone marrow stromal cells (hBMSCs). hBMSCs were injected intravenously into EAE mice upon onset of paresis. Neurological functional tests were scored daily by grading clinical signs (score 0-5). Immunohistochemistry was performed to measure the transplanted hBMSCs, cell proliferation (bromodeoxyuridine, BrdU), oligodendrocyte progenitor cells (NG2), oligodendrocytes (RIP), and brain-derived neurotrophic factor (BDNF). The maximum clinical score and the average clinical scores were significantly decreased in the hBMSC-transplanted mice compared to the phosphate-buffered-saline-treated EAE controls, indicating a significant improvement in function. Demyelination significantly decreased, and BrdU(+) and BDNF(+) cells significantly increased in the hBMSC-treated mice compared to controls. Some BrdU(+) cells were colocalized with NG2(+) and RIP(+) immunostaining. hBMSCs also significantly reduced the numbers of vessels containing inflammatory cell infiltration. These data indicate that hBMSC treatment improved functional recovery after EAE in mice, possibly, via reducing inflammatory infiltrates and demyelination areas, stimulating oligodendrogenesis, and by elevating BDNF expression.

Erythropoietin treatment improves neurological functional recovery in EAE mice.

Erythropoietin (EPO), originally recognized for its central role in erythropoiesis, has been shown to improve neurological outcome after stroke. Here, we investigated the treatment of experimental autoimmune encephalomyelitis (EAE) in mice with EPO. Mice were treated with recombinant human EPO (rhEPO) upon onset of paresis. Neurological functional tests were scored daily by grading of clinical signs (score 0-5). Hematoxylin and eosin (HE) staining of cerebral tissue was performed to detect inflammatory infiltrates. Double staining for Luxol fast blue and Bielshowsky was used to demonstrate myelin and axons, respectively. Immunohistochemistry was performed to measure the expression of bromodeoxyuridine (BrdU, a marker for cell proliferation), NG2 (a marker for oligodendrocyte progenitor cells) and brain-derived neurotrophic factor (BDNF). Treatment with rhEPO significantly improved neurological functional recovery, reduced inflammatory infiltrates and demyelination, and increased oligodendrocyte progenitor cell proliferation and BDNF+ cells compared to the EAE controls. These data indicate that rhEPO treatment improved functional recovery after EAE in mice, possibly, via stimulating oligodendrogenesis, downregulating proinflammatory infiltrates and by elevating BDNF expression.