Experimental Autoimmune Encephalomyelitis Rats Research Articles

Ibudilast produces anti-allodynic effects at the persistent phase of peripheral or central neuropathic pain in rats: Different inhibitory mechanism on spinal microglia from minocycline and propentofylline

Microglia exhibit various activation phenotypes in the spinal cord after peripheral nerve injury, and promote neuropathic pain. Ibudilast is a phosphodiesterase inhibitor with anti-inflammatory activity, but its effect on activated microglia in chronic neuropathic pain is poorly understood. We investigated whether ibudilast was effective on established allodynia associated with activated microglial phenotypes in two rat models of peripheral and central neuropathic pain. A single intrathecal injection of ibudilast (25 μg) inhibited established allodynia on days 7–21 after sciatic nerve injury in rats. Repeated injections of ibudilast (25 μg/day) reduced the numbers of phosphorylated p38-positive cells without changing hypertrophic microglia, whereas minocycline (100 μg/day) decreased the numbers of hypertrophic microglia associated with phosphorylated p38 levels in the spinal cord. Gene analysis revealed that minocycline, but not ibudilast, increased the expression of anti-inflammatory cytokine genes Il10 and Tgfβ1 in the spinal cord. Propentofylline (100 μg/day) was less effective on microglial phenotypes and established allodynia. Ibudilast inhibited persistent allodynia after the recovery of motor deficits in experimental autoimmune encephalomyelitis rats. Therefore, ibudilast might be effective for chronic neuropathic pain after peripheral and central nerve damage. Ibudilast mediated these effects on activated microglia using a different mechanism compared with minocycline and propentofylline.

The possible anti-apoptotic and antioxidant effects of acetyl l-carnitine as an add-on therapy on a relapsing-remitting model of experimental autoimmune encephalomyelitis in rats

Multiple sclerosis (MS) is a progressive inflammatory autoimmune demyelinating disease of the brain and spinal cord. Glucocorticoids (GCs) are the standard treatment of MS, however they have several drawbacks like oxidative stress and apoptosis. This study was designed to evaluate some possible antioxidant, anti-apoptotic and immune modulatory effects of Acetyl-l-carnitine (ALCAR) when used either alone or as an add-on therapy with dexamethasone for treatment of a relapsing-remitting (RR) experimental autoimmune encephalomyelitis (EAE) as a model of MS. This experiment was performed on 50 female Sprague Dawley rats divided into; normal control group, untreated EAE group, EAE group treated by dexamethasone, EAE group treated by ALCAR, and EAE group treated by both dexamethasone and ALCAR. The clinical score of the motor deficit of EAE was recorded daily. At the end of experiment, rats were sacrificed and the brain and spinal cord were processed for assessment of reduced glutathione (GSH), malondialdehyde (MDA) and caspase-3 activity. Histopathological changes and immunohistochemical expression of Bcl-2 and CD4+ T cell were carried out. Combination of both dexamethasone and ALCAR provided marked antioxidant and anti-apoptotic effects represented by significant decrease in MDA, caspase-3 and significant increase in GSH, Bcl-2 expression, and it also exhibited marked immunosuppressive effect represented by significant decrease in CD4+ T cells expression with significant improvement in clinical outcome when compared to untreated EAE group or to dexamethasone treated group. These findings pave the way for using ALCAR as an adjuvant therapy during long-term use of dexamethasone in MS.

Gut Microbiota Confers Resistance of Albino Oxford Rats to the Induction of Experimental Autoimmune Encephalomyelitis.

Albino Oxford (AO) rats are extremely resistant to induction of experimental autoimmune encephalomyelitis (EAE). EAE is an animal model of multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS), with established autoimmune pathogenesis. The autoimmune response against the antigens of the CNS is initiated in the peripheral lymphoid tissues after immunization of AO rats with CNS antigens. Subsequently, limited infiltration of the CNS occurs, yet without clinical sequels. It has recently become increasingly appreciated that gut-associated lymphoid tissues (GALT) and gut microbiota play an important role in regulation and propagation of encephalitogenic immune response. Therefore, modulation of AO gut microbiota by antibiotics was performed in this study. The treatment altered composition of gut microbiota in AO rats and led to a reduction in the proportion of regulatory T cells in Peyer’s patches, mesenteric lymph nodes, and in lymph nodes draining the site of immunization. Upregulation of interferon-γ and interleukin (IL)-17 production was observed in the draining lymph nodes. The treatment led to clinically manifested EAE in AO rats with more numerous infiltrates and higher production of IL-17 observed in the CNS. Importantly, transfer of AO gut microbiota into EAE-prone Dark Agouti rats ameliorated the disease. These results clearly imply that gut microbiota is an important factor in AO rat resistance to EAE and that gut microbiota transfer is an efficacious way to treat CNS autoimmunity. These findings also support the idea that gut microbiota modulation has a potential as a future treatment of multiple sclerosis.

Characterization of Transglutaminase 2 activity inhibitors in monocytes in vitro and their effect in a mouse model for multiple sclerosis.

The neurodegenerative disease multiple sclerosis (MS) is pathologically characterized by the massive influx of immune cells into the central nervous system. This contributes to demyelination and axonal damage which causes symptoms such as motor and cognitive dysfunctions. The migration of leukocytes from the blood vessel is orchestrated by a multitude of factors whose determination is essential in reducing cellular influx in MS patients and the experimental autoimmune encephalomyelitis (EAE) animal model. The here studied enzyme tissue Transglutaminase (TG2) is present intracellularly, on the cell surface and extracellularly. There it contributes to cellular adhesion and migration via its transamidation activity and possibly by facilitating cellular interaction with the extracellular matrix. Previous data from our group showed reduced motor symptoms and cellular infiltration after using a pharmacological TG2 transamidation activity inhibitor in a rat EAE model. However, it remained elusive if the cross-linking activity of the enzyme resulted in the observed effects. To follow-up, we now characterized two new small molecule TG2 activity inhibitors, BJJF078 and ERW1041E. Both compounds are potent inhibitor of recombinant human and mouse Transglutaminase enzyme activity, mainly TG2 and the close related enzyme TG1. In addition they did not affect the binding of TG2 to the extracellular matrix substrate fibronectin, a process via which TG2 promotes cellular adhesion and migration. We found, that ERW1041E but not BJJF078 resulted in reduced EAE disease motor-symptoms while neither caused apparent changes in pathology (cellular influx), Transglutaminase activity or expression of inflammation related markers in the spinal cord, compared to vehicle treated controls. Although we cannot exclude issues on bioavailability and in vivo efficacy of the used compounds, we hypothesize that extracellular TG1/TG2 activity is of greater importance than (intra-)cellular activity in mouse EAE pathology.

A Kv1.3 channel-specific blocker alleviates neurological impairment through inhibiting T-cell activation in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neurological autoimmune disorder characterized by mistaken attacks of inflammatory cells against the central nervous system (CNS), resulting in demyelination and axonal damage. Kv1.3 channel blockers can inhibit T-cell activation and have been designed for MS therapy. However, little is known about the effects of Kv1.3 blockers on protecting myelin sheaths/axons in MS. This study aimed at investigating the neuroprotection efficacy of a selective Kv1.3 channel blocker ImKTx88 (ImK) in MS animal model. Experimental autoimmune encephalomyelitis (EAE) rat model was established. The neuroprotective effect of ImK was assessed by immunohistochemistry and transmission electron microscopy (TEM). In addition, the antiinflammatory effect of ImK by suppressing T-cell activation was assessed by flow cytometry and ELISA in vitro. Our results demonstrated that ImK administration ameliorated EAE clinical severity. Moreover, ImK increased oligodendrocytes survival, preserved axons, and myelin integrity and reduced the infiltration of activated T cells into the CNS. This protective effect of the peptide may be related to its suppression of autoantigen-specific T-cell activation via calcium influx inhibition. ImK prevents neurological damage by suppressing T-cell activation, suggesting the applicability of this peptide in MS therapy.

Olfactory Dysfunction in Autoimmune Central Nervous System Neuroinflammation.

Olfactory dysfunction is an early sign of neuroinflammation of the central nervous system (CNS). Microgliosis and astrogliosis are representative pathological changes that develop during neuroinflammation of CNS tissues. Autoimmune CNS inflammation, including human multiple sclerosis, is an occasional cause of olfactory disorders. We evaluated whether gliosis and olfactory dysfunction developed in animals with experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Neuroinflammatory lesions characterized by infiltration of inflammatory cells and microglial cell activation were occasionally found in the olfactory bulbs of EAE-affected rats. Microglial activation, visualized by immunohistochemical staining of ionized calcium binding protein (Iba)-1, and astrogliosis in the olfactory bulb were also evident in the olfactory bulb of EAE rats. Inflammatory cells were found along the olfactory nerves and in the olfactory submucosa. Western blot analysis of olfactory marker protein (OMP) levels showed that OMP expression was significantly downregulated in the olfactory mucosa of EAE rats. On the buried food test, EAE-affected mice required significantly more time to find a bait pellet. Collectively, the results suggest that the olfactory dysfunction of EAE is closely linked to downregulation of OMP and the development of inflammatory foci in the olfactory system in an animal model of human multiple sclerosis.

A Peptide Targeting Inflammatory CNS Lesions in the EAE Rat Model of Multiple Sclerosis.

Multiple sclerosis is characterized by inflammatory lesions dispersed throughout the central nervous system (CNS) leading to severe neurological handicap. Demyelination, axonal damage, and blood brain barrier alterations are hallmarks of this pathology, whose precise processes are not fully understood. In the experimental autoimmune encephalomyelitis (EAE) rat model that mimics many features of human multiple sclerosis, the phage display strategy was applied to select peptide ligands targeting inflammatory sites in CNS. Due to the large diversity of sequences after phage display selection, a bioinformatics procedure called "PepTeam" designed to identify peptides mimicking naturally occurring proteins was used, with the goal to predict peptides that were not background noise. We identified a circular peptide CLSTASNSC called "Ph48" as an efficient binder of inflammatory regions of EAE CNS sections including small inflammatory lesions of both white and gray matter. Tested on human brain endothelial cells hCMEC/D3, Ph48 was able to bind efficiently when these cells were activated with IL1β to mimic inflammatory conditions. The peptide is therefore a candidate for further analyses of the molecular alterations in inflammatory lesions.

Effect of matrine on Calpain/MAP-2 of experimental autoimmune encephalomyelitic rats

Objective To study the effect of matrine on Calpain and MAP-2 in rats with experimental autoimmune encephalomyelitis(EAE). Methods In accordance with the random number table, 60 Wistar rats were divided into 6 groups randomly: normal group, model group, dexamethasone(DEX)-treated group(1mg/kg), high-dose matrine(MAT)-treated group(250mg/kg), middle-dose MAT-treated group(200mg/kg) and low-dose MAT-treated group(150mg/kg). The EAE models were induced by immunized spinal cord extracts of guinea pig with complete Freunds' adjuvant.Rats of three MAT-treated groups and DEX-treated group were injected intraperitoneally with MAT and DEX daily for 16 days respectively, whereas rats of normal group and model group were injected intraperitoneally with normal saline.Clinical signs of rats in six groups were observed daily.Hematoxylin-eosin(HE) was used to analyze histopathological evaluation of spinal cord.μ-Calpain, m-Calpain and MAP-2 in spinal cord were determined using RT-PCR and immunohistochemistry respectively. Results Compared with the model group[(2.85±0.78)points], the clinical scores were significantly decreased in high-dose-MAT group[(1.28±0.59)points], middle-dose-MAT group[(1.45±0.64)points] and low-dose-MAT group[(2.09±0.71)points](t=5.345, 4.314, 2.869, all P<0.05). The HE score of rats in model group[(2.49±0.29)points] was significantly higher than that in high-dose-MAT group[(1.04±0.26)points], middle-dose-MAT group[(1.29±0.20)points] and low-dose-MAT group[(1.77±0.24)points](t=5.185, 4.274, 3.629, all P<0.01). The levels of μ-Calpain mRNA and m-Calpain mRNA in the three MAT-treated groups were significantly lower than those in model group(t=10.656, 9.418, 7.044, all P<0.01; t=6.332, 5.416, 3.978, all P<0.01). In addition, the expression of MAP-2 in the spinal cord of EAE rats showed a marked elevation after MAT treatment(t=12.841, 9.924, 7.038, all P<0.01). Conclusion Matrine may be an effective therapeutic approach for EAE by inhibiting Calpain and increase MAP-2 expression. Key words: Matrine; Encephalomyelitis, autoimmune, experimental; Calpain; Microtubule associated protein-2; Rats

A novel PADRE-Kv1.3 vaccine effectively induces therapeutic antibodies and ameliorates experimental autoimmune encephalomyelitis in rats

Previous studies have confirmed that selective blockade of Kv1.3 channels could modulate the activities of pathogenic T cells and microglia/macrophages, which play key roles in experimental autoimmune encephalomyelitis (EAE). In this study, we designed an anti-Kv1.3 vaccine (PADRE-Kv1.3) to explore its protective role in EAE rat models. When the vaccine was applied in EAE rats, clinical scores and several staining techniques were used to evaluate the severity of the disease. T cell subtypes and related cytokines, as well as microglia/macrophage activation were assayed through flow cytometry, qRT-PCR or immunofluorescence staining, respectively. We herein showed that rats and mice developed high titers of anti-Kv1.3 antibodies and appeared no abnormal manifestations after the PADRE-Kv1.3 vaccine treatment. In EAE models, the vaccine treatment effectively alleviated the clinical severity and lessened pathological damages in the central nervous system (CNS). In addition, we found the vaccine significantly decreased the number of pathogenic T cells (Th17 and IFN-γ–producing T cells) and the production of related pro-inflammatory cytokines (IL-17A, IFN-γ and IL-1β), but increased the number of protective T subsets (CD4+IL-10+ T cells and Treg cells) in the spleen or CNS. Moreover, the infiltration of microglia/macrophages significantly reduced and these cells shifted toward anti-inflammatory M2 subtype in the CNS after the vaccine treatment. Thus, we demonstrated that the PADRE-Kv1.3 vaccine could induce therapeutic anti-Kv1.3 antibodies and ameliorate EAE in rats effectively and safely, which provides a new field of vision for the protection and therapy of multiple sclerosis.

Cholinergic imbalance in lumbar spinal cord of a rat model of multiple sclerosis

Cholinergic dysfunction in the central nervous system is an important characteristic of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). By using a rat EAE model, upregulation of vesicular acetylcholine transporter (VAChT) level in the EAE rat lumbar spinal cord was detected by western blot and immunostaining, and was associated with lymphocyte filtration and glial activation. Ex vivo and in vitro autoradiography studies with [18F]VAT, a VAChT-specific radioligand, also revealed increased tracer uptake in EAE rat lumbar spinal cord compared with shams. These studies on VAChT expression suggest central cholinergic imbalance during EAE progression.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

In this report, amide-linked cyclic peptide analogues of the 87–99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87–99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72–85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87–99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91–99)[Ala96]MBP87–99, cyclo(87–99)[Ala91,96]MBP87–99 and cyclo(87–99)[Arg91, Ala96]MBP87–99, but not wild-type linear MBP87–99, strongly inhibited MBP72–85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87–99)[Arg91, Ala96]MBP87–99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Therapeutic Potential of Pien Tze Huang on Experimental Autoimmune Encephalomyelitis Rat.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). There is still lack of commercially viable treatment currently. Pien Tze Huang (PZH), a traditional Chinese medicine, has been proved to have anti-inflammatory, neuroprotective, and immunoregulatory effects. This study investigated the possible therapeutic effects of PZH on experimental autoimmune encephalomyelitis (EAE) rats, a classic animal model of MS. Male Lewis rats were immunized with myelin basic protein (MBP) peptide to establish an EAE model and then treated with three doses of PZH. Clinical symptoms, organ coefficient, histopathological features, levels of proinflammatory cytokines, and chemokines as well as MBP and Olig2 were analyzed. The results indicated that PZH ameliorated the clinical severity of EAE rats. It also remarkably reduced inflammatory cell infiltration in the CNS of EAE rats. Furthermore, the levels of IL-17A, IL-23, CCL3, and CCL5 in serum and the CNS were significantly decreased; the p-P65 and p-STAT3 levels were also downregulated in the CNS, while MBP and Olig2 in the CNS of EAE rats had a distinct improvement after PZH treatment. In addition, PZH has no obvious toxicity at the concentration of 0.486 g/kg/d. This study demonstrated that PZH could be used to treat MS.

Amelioration of experimental autoimmune encephalomyelitis by Ishige okamurae.

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune central nervous system disease characterized by inflammation with oxidative stress. The aim of this study was to evaluate an anti-inflammatory effect of Ishige okamurae on EAE-induced paralysis in rats. An ethanolic extract of I. okamurae significantly delayed the first onset and reduced the duration and severity of hind-limb paralysis. The neuropathological and immunohistochemical findings in the spinal cord were in agreement with these clinical results. T-cell proliferation assay revealed that the ethyl-acetate fraction of I. okamurae suppressed the proliferation of myelin basic protein reactive T cells from EAE affected rats. Flow cytometric analysis showed TCRαβ+ T cells was significantly reduced in the spleen of EAE rats with I. okamurae treatment with concurrent decrease of inflammatory mediators including tumor necrosis factor-α and cyclooxygenase-2. Collectively, it is postulated that I. okamurae ameliorates EAE paralysis with suppression of T-cell proliferation as well as decrease of pro-inflammatory mediators as far as rat EAE is concerned.

Valproic acid attenuates inflammation of optic nerve and apoptosis of retinal ganglion cells in a rat model of optic neuritis

AimsOptic neuritis (ON) is an inflammatory disease of the optic nerve, which often occurs in patients with multiple sclerosis (MS) and leads to retinal ganglion cell (RGC) death and even severe visual loss. Valproic acid (VPA) is a short-chain branched fatty acid with anti-epileptic, neuro-protective and anti-inflammatory effects. Here, we examined the effects of VPA in experimental autoimmune encephalomyelitis (EAE) rats and explored the underlying mechanisms. Main methodsEAE was induced by subcutaneous injection with myelin basic protein, emulsified with complete Freund’s adjuvant and Mycobacterium tuberculosis H37Ra into the Lewis rats. Subsequently, animals in the VPA groups were treated orally with VPA (250 or 500 mg/kg) once a day for 13 days. Key findingsVPA treatment significantly attenuated inflammation and microgliosis in optic nerve in EAE-ON rats, as evidenced by the decrease in the mRNA levels of interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-17, and inducible nitric oxide synthase (iNOS), the suppression in nuclear factor (NF)-κB signal pathway as well as the down-regulation of CD11b expression in optic nerve. Additionally, the apoptotic RGCs were remarkably increased in the EAE retina, which was inhibited by VPA treatment. Consistent with the TUNEL staining, VPA administration also obviously suppressed the ratio of Bax: Bcl-2 and the expression of cleaved caspase-3 and PARP in optic nerve in EAE rats. SignificanceOur findings demonstrated that VPA treatment could prevent inflammation responses and RGC apoptosis in optic nerve in EAE-ON rats, suggesting that VPA may be available for optic nerve protection during ON.

Purinergic receptors P2Y12R and P2X7R: potential targets for PET imaging of microglia phenotypes in multiple sclerosis

BackgroundMicroglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression. The activation status of microglia in vivo is highly dynamic and occurs as a continuum, with the pro-inflammatory and anti-inflammatory phenotypes on either end of this spectrum. Little is known about in vivo dynamics of microglia phenotypes in MS due to the lack of diagnostic tools. Positron emission tomography (PET) imaging is a powerful non-invasive technique that allows real-time imaging of microglia activation phenotypes in the central nervous system, depending on the availability of selective PET tracers. Our objective is to investigate and characterize the expression of the purinergic receptors P2Y12R and P2X7R as potential targets for PET tracer development and subsequent PET imaging in order to evaluate the dynamics of microglia status in vivo.MethodsWe used immunohistochemical analysis to explore the expression of P2Y12R and P2X7R in experimental autoimmune encephalomyelitis (EAE) post-mortem tissues and different stages of well-characterized MS lesions. We evaluated by quantitative real-time polymerase chain reaction the expression of P2Y12R and P2X7R in human polarized microglia, and we performed autoradiography binding assay with radiolabeled P2Y12R and P2X7R antagonists using MS and rat EAE tissues.ResultsHere, we demonstrate that P2X7R is associated with a pro-inflammatory phenotype of human microglia in vitro, and is highly expressed in microglia in MS lesions as well as during the peak of EAE. In contrast, P2Y12R was associated with an anti-inflammatory phenotype in human microglia in vitro and was expressed at lower levels in active inflammatory MS lesions compared to normal-appearing white matter (NAWM) and similarly in EAE, while its expression increased in the remission phase of EAE. Binding of radiolabeled tracers specific for P2Y12R and P2X7R on ex vivo tissues validated the value of these receptors as PET imaging targets for microglia phenotypes in vivo.ConclusionOur results suggest that P2Y12R and P2X7R are excellent targets for PET imaging to discriminate distinct microglia phenotypes in MS. Ultimately, this may provide insight into the role of microglia in disease progression and monitor novel treatment strategies to alter microglia phenotype.

Corticosteroid therapy exacerbates the reduction of melatonin in multiple sclerosis

ObjectivesCorticosteroid therapy is employed in multiple sclerosis (MS), a neurological abnormality characterized by an inflammatory process. Melatonin, a potent sleep-promoting and circadian phase regulatory hormone, is produced mainly in the pineal gland whose inhibition leads to sleep disturbances. MethodsIn this study, methylprednisolone (MP) corticosteroid treatment was used in an acute experimental autoimmune encephalomyelitis (EAE) rat model (intraperitoneal, 30 mg/kg) and in MS patients (intravenous, 1000 mg/day), followed by assessing melatonin serum levels. Key findingsResults showed that mean clinical scores were significantly improved in MP- versus PBS-treated EAE rats (1.5 vs 4.1, respectively). In addition, MP was found to induce a significant decrease in serum IFN-γ, whereas IL-4 levels were significantly increased, in comparison to PBS-treated EAE rats. The ratio of IFN-γ/IL-4, which acts as an indicator of Th-1/Th-2, was significantly lower in MP treated, compared to PBS treated EAE rats or controls. Moreover, serum levels of melatonin showed a significant decrease in the MP group, compared to normal rats. Moreover, MP therapy for 1 or 2 days resulted in a significant reduction of melatonin serum levels in MS patients. ConclusionsSince corticosteroids cause a reduction in melatonin serum levels, an important hormone in sleep regulation, their prescription to MS patients should be carefully considered. Corticosteroids could be a cause of insomnia and sleep disturbance in patients receiving this type of medication.

Fluvoxamine stimulates oligodendrogenesis of cultured neural stem cells and attenuates inflammation and demyelination in an animal model of multiple sclerosis

Multiple Sclerosis (MS) require medications controlling severity of the pathology and depression, affecting more than half of the patients. In this study, the effect of antidepressant drug fluvoxamine, a selective serotonin reuptake inhibitor, was investigated in vitro and in vivo. Nanomolar concentrations of fluvoxamine significantly increased cell viability and proliferation of neural stem cells (NSCs) through increasing mRNA expression of Notch1, Hes1 and Ki-67, and protein levels of NICD. Also, physiological concentrations of fluvoxamine were optimal for NSC differentiation toward oligodendrocytes, astrocytes and neurons. In addition, fluvoxamine attenuated experimental autoimmune encephalomyelitis (EAE) severity, a rat MS model, by significantly decreasing its clinical scores. Moreover, fluvoxamine treated EAE rats showed a decrease in IFN-γ serum levels and an increase in IL-4, pro- and anti-inflammatory cytokines respectively, compared to untreated EAE rats. Furthermore, immune cell infiltration and demyelination plaque significantly decreased in spinal cords of fluvoxamine-treated rats, which was accompanied by an increase in protein expression of MBP and GFAP positive cells and a decrease in lactate serum levels, a new biomarker of MS progression. In summary, besides its antidepressant activity, fluvoxamine stimulates proliferation and differentiation of NSCs particularly toward oligodendrocytes, a producer of CNS myelin.

Kv1.3 channel blocker (ImKTx88) maintains blood\u2013brain barrier in experimental autoimmune encephalomyelitis

BackgroundDisruption of blood–brain barrier (BBB) and subsequent infiltration of auto-reactive T lymphocytes are major characteristics of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Kv1.3 channel blockers are demonstrated potential therapeutic effects on MS patients and EAE models, maybe via reducing activation of T cells. However, it remains to be explored whether Kv1.3 channel blockers maintain integrity of BBB in MS model.ResultsIn this study, ImKTx88, a highly selective Kv1.3 channel blocker, was used to determine the role of Kv1.3 channel in the pathogenesis of EAE, particularly in the maintenance of BBB. ImKTx88 ameliorated pathological severity in the EAE rats, and reduced extravasation into CNS. ImKTx88 also ameliorated the severity of loss or redistribution of tight junction proteins, and inhibited over-expression of ICAM-1 and VCAM-1 in the brain from EAE rats. Furthermore ImKTx88 protection was associated with activation of Ang-1/Tie-2 axis, and might be due to decreased IL-17 production.ConclusionsImKTx88 may be a novel therapeutic agent for MS treatment by stabilizing the BBB.

Sphingosine Toxicity in EAE and MS: Evidence for Ceramide Generation via Serine-Palmitoyltransferase Activation.

Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.

Pharmacologic characterizations of a P2X7 receptor-specific radioligand, [11C]GSK1482160 for neuroinflammatory response.

The P2X7 receptor (P2X7R) is a key regulatory element in the neuroinflammatory cascade that provides a promising target for imaging neuroinflammation. GSK1482160, a P2X7R modulator with nanomolar binding affinity and high selectivity, has been successfully radiolabeled and utilized for imaging P2X7 levels in a mouse model of lipopolysaccharide-induced systemic inflammation. In the current study, we further characterized its binding profile and determined whether [C]GSK1482160 can detect changes in P2X7R expression in a rodent model of multiple sclerosis. [C]GSK1482160 was synthesized with high specific activity and high radiochemical purity. Radioligand saturation and competition binding assays were performed for [C]GSK1482160 using HEK293-hP2X7R living cells. Micro-PET studies were carried out in nonhuman primates. In vitro autoradiography and immunohistochemistry studies were then carried out to evaluate tracer uptake and P2X7 expression in experimental autoimmune encephalomyelitis (EAE) rat lumbar spinal cord at EAE-peak and EAE-remitting stages compared with sham rats. [C]GSK1482160 binds to HEK293-hP2X7R living cells with high binding affinity (Kd=5.09±0.98 nmol/l, Ki=2.63±0.6 nmol/l). Micro-PET studies showed high tracer retention and a homogeneous distribution in the brain of nonhuman primates. In the EAE rat model, tracer uptake of [C]GSK1482160 in rat lumbar spinal cord was the highest at the EAE-peak stage (277.74±79.74 PSL/mm), followed by the EAE-remitting stage(149.00±54.14 PSL/mm) and sham (66.37±1.48 PSL/mm). The tracer uptake correlated strongly with P2X7-positive cell counts, activated microglia numbers, and disease severity. We conclude that [C]GSK1482160 has the potential for application in monitoring neuroinflammation.