Bao-Guo Xiao

Targeting the Shift from M1 to M2 Macrophages in Experimental Autoimmune Encephalomyelitis Mice Treated with Fasudil

We observed the therapeutic effect of Fasudil and explored its mechanisms in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Fasudil, a selective Rho kinase (ROCK) inhibitor, was injected intraperitoneally at 40 mg/kg/d in early and late stages of EAE induction. Fasudil ameliorated the clinical severity of EAE at different stages, and decreased the expression of ROCK-II in spleen, accompanied by an improvement in demyelination and inhibition of inflammatory cells. Fasudil mainly inhibited CD4+IL-17+ T cells in early treatment, but also elevated CD4+IL-10+ regulatory T cells and IL-10 production in late treatment. The treatment of Fasudil shifted inflammatory M1 to anti-inflammatory M2 macrophages in both early and late treatment, being shown by inhibiting CD16/32, iNOS, IL-12, TLR4 and CD40 and increasing CD206, Arg-1, IL-10 and CD14 in spleen. By using Western blot and immunohistochemistry, iNOS and Arg-1, as two most specific markers for M1 and M2, was inhibited or induced in splenic macrophages and spinal cords of EAE mice treated with Fasudil. In vitro experiments also indicate that Fasudil shifts M1 to M2 phenotype, which does not require the participation or auxiliary of other cells. The polarization of M2 macrophages was associated with the decrease of inflammatory cytokine IL-1β, TNF-α and MCP-1. These results demonstrate that Fasudil has therapeutic potential in EAE possibly through inducing the polarization of M2 macrophages and inhibiting inflammatory responses.

Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice.

J. Neurochem. (2010) 114, 1619–1629.

Fasudil, a Rho kinase inhibitor, drives mobilization of adult neural stem cells after hypoxia/reoxygenation injury in mice.

Rho kinase (ROCK) is important in fundamental processes of cell proliferation and survival. Blockade of ROCK promotes stem cell survival in vitro and axonal regeneration in vivo, exhibiting therapeutic potential such as spinal cord injuries and stroke. Here, we used the model of hypoxia/reoxygenation (H/R) injury to explore the possibility whether Fasudil, a ROCK inhibitor in clinical application for subarachnoid hemorrhage and stroke, mobilizes adult neural stem cells in vivo. Most interestingly, Fasudil triggers neurogenesis especially in the subventricular zone after H/R. The increase of Brdu+ cholinergic neurons was observed in striatum and forebrain cortex of Fasudil-treated mice after 30 days. Further observation demonstrates that both levels of granulocyte colony-stimulating factor (G-CSF) and astrocytes expressing G-CSF were elevated in mice treated with Fasudil, as compared to mice injected with saline. In vitro H/R model of cultured astrocytes, Fasudil promoted astrocytes to produce G-CSF in a dose-dependent manner. In addition, antibody neutralization and receptor blocking of the G-CSF pathway clearly demonstrate that Fasudil-induced neurogenesis was mediated partially through astrocyte-derived G-CSF. Our results indicate that Fasudil might represent a promising therapeutic perspective by mobilizating endogenous adult neural stem cells in the CNS.

A putative mechanism on remission of multiple sclerosis during pregnancy: estrogen-induced indoleamine 2,3-dioxygenase by dendritic cells

The basis for the reduced relapse rate of multiple sclerosis (MS) during pregnancy remains unexplained but, if defined, could create novel treatment options. Estrogen constitutes one candidate molecule, but the mechanism by which estrogen may affect MS during pregnancy is unclear. In this study, we used monocyte-derived dendritic cells (DCs) from MS patients to explore the estrogen (17-b-estradiol)-related pathway of immune modulation. Estrogen induced the expression of indoleamine 2,3-dioxygenase (IDO) on DCs, limiting T-cell proliferation and both Th1 and Th2 cytokine production. The suppression of T-cell proliferation mediated by estrogenexposed DCs was partly abolished by the IDO-inhibitor, 1-methyl-dl-tryptophan, indicating that estrogen-exposed DCs induced IDO-dependent T-cell suppression. Our data support the hypothesis that the change in the clinical course of MS observed in pregnancy may be related to the estrogen DC-IDO axis, which could represent a novel target for MS therapy.

Comparative Evaluation of Early Diagnosis of Tuberculous Meningitis by Different Assays

ABSTRACT Cerebrospinal fluid (CSF) and peripheral blood (PBL) were sampled multiple times from 25 patients with a clinical diagnosis of tuberculous meningitis (TBM) and 49 controls, including 27 patients with other infectious diseases of the central nervous system and 22 patients with other noninfectious neurological diseases. We used an enzyme-linked immunospot assay (ELISPOT) to detect anti-Mycobacterium bovis BCG antibody-secreting cells in CSF and PBL, PCR to detect a repeated insertion sequence (IS6110) specific for Mycobacterium tuberculosis in CSF, and an enzyme-linked immunosorbent assay (ELISA) to detect anti-BCG antibodies in CSF and PBL. In the meantime, culture of CSF from every TBM and control patient was done on Lowenstein-Jensen medium. ELISPOT proved to be the most valuable test, with a sensitivity of 84.0% and a specificity of 91.8%, and showed a sensitivity of 100.0% with the CSF specimens obtained within 4 weeks after the onset of TBM. The numbers of CSF anti-BCG immunoglobulin-secreting cells tested by ELISPOT were even higher in the early phase of TBM and declined while the disease was going on (P = 0.008), which allowed an early diagnosis to be made. The sensitivities of PCR and ELISA were only 75.0% and 52.3%, respectively; and the specificities were 93.7% and 91.6%, respectively. Culture of CSF on Lowenstein-Jensen medium was the least sensitive (16%) compared to the sensitivities of the other three assays. Our results demonstrate that the ELISPOT technique is worthy for routine use in the laboratory to support the clinical diagnosis of TBM.

Rho kinase inhibitor Fasudil induces neuroprotection and neurogenesis partially through astrocyte-derived G-CSF.

Rho-kinases (ROCK) are serine/threonine kinases that play an important role in fundamental processes of cell migration, proliferation and survival. Blockade of ROCK promotes axonal regeneration and neuroprotection, thereby exhibiting therapeutic potentials for clinical application to spinal cord damage and stroke. Here we explored the mechanisms of Fasudil, a ROCK inhibitor, in neuroprotection and neurogenesis by using oxygen-glucose deprivation (OGD) as an in vitro ischemia model. Fasudil stimulates astrocytes to produce granulocyte colony-stimulating factor (G-CSF). Astrocyte-conditioned medium treated with Fasudil (ACM-F) contributes to the generation of neurospheres, and decreases neuron death. Neutralization of G-CSF in ACM-F and blocking of G-CSF receptor in neuronal cell cultures revealed that Fasudil-induced neuroprotection and/or neurogenesis are mediated partially through astrocyte-derived G-CSF. Our results indicate that ROCK inhibition by Fasudil, protecting neurons and mobilizating neural stem cells, might represent a useful therapeutic perspective for various neurological disorders characterized by neuron death.

Treatment with Trehalose Prevents Behavioral and Neurochemical Deficits Produced in an AAV α-Synuclein Rat Model of Parkinson’s Disease

The accumulation of misfolded α-synuclein in dopamine (DA) neurons is believed to be of major importance in the pathogenesis of Parkinson’s disease (PD). Animal models of PD, based on viral-vector-mediated over-expression of α-synuclein, have been developed and show evidence of dopaminergic toxicity, providing us a good tool to investigate potential therapies to interfere with α-synuclein-mediated pathology. An efficient disease-modifying therapeutic molecule should be able to interfere with the neurotoxicity of α-synuclein aggregation. Our study highlighted the ability of an autophagy enhancer, trehalose (at concentrations of 5 and 2xa0% in drinking water), to protect against A53T α-synuclein-mediated DA degeneration in an adeno-associated virus serotype 1/2 (AAV1/2)-based rat model of PD. Behavioral tests and neurochemical analysis demonstrated a significant attenuation in α-synuclein-mediated deficits in motor asymmetry and DA neurodegeneration including impaired DA neuronal survival and DA turnover, as well as α-synuclein accumulation and aggregation in the nigrostriatal system by commencing 5 and 2xa0% trehalose at the same time as delivery of AAV. Trehalose (0.5xa0%) was ineffective on the above behavioral and neurochemical deficits. Further investigation showed that trehalose enhanced autophagy in the striatum by increasing formation of LC3-II. This study supports the concept of using trehalose as a novel therapeutic strategy that might prevent/reverse α-synuclein aggregation for the treatment of PD.

Impaired regulatory function and enhanced intrathecal activation of B cells in neuromyelitis optica: distinct from multiple sclerosis

Background: The effective treatment of neuromyelitis optica (NMO) with rituximab has suggested an important role for B cells in NMO pathogenesis. Objective: To explore the antibody-independent function of B cells in NMO and relapsing–remitting multiple sclerosis (RRMS). Methods: Fifty-one NMO patients and 42 RRMS patients in an acute relapse phase and 37 healthy controls (HC) were enrolled in the study. The B cell expression of B cell activating factor receptor (BAFF-R), CXCR5 and very late antigen-4 (VLA-4), the B cell production of interleukin (IL)-10 and interferon (IFN)-γ and the proportion of circulating memory and CD19+CD24highCD38high regulatory B cells were evaluated by flow cytometry. The cerebrospinal fluid (CSF) levels of BAFF and CXCL13 were determined by enzyme-linked immunosorbent assay (ELISA). Results: The CD19+CD24highCD38high regulatory B cell levels and the B cell expression of IL-10 were significantly lower in NMO patients than in RRMS patients and the HC. In aquaporin-4 antibody (AQP4-ab)-positive NMO patients, the B cell IL-10 production and CD19+CD24highCD38high regulatory B cell levels were even lower than in AQP4-ab-negative NMO patients. The CSF BAFF and CXCL13 levels were significantly higher in NMO patients than in patients with RRMS and other non-inflammatory neurologic diseases (ONDs). Conclusions: The immuno-regulatory properties of B cells are significantly impaired in NMO patients and particularly in AQP4-ab-positive NMO patients. The elevated CSF levels of BAFF and CXCL13 in NMO suggest an enhanced intrathecal B cell recruitment and activation. Our results further define the distinct immunological nature of NMO and RRMS from the B cell perspective.

Inhibition of caspase-8 attenuates neuronal death induced by limbic seizures in a cytochrome c-dependent and Smac/DIABLO-independent way.

There is increasing evidence that neuronal cell death induced by seizures occurs via extrinsic (death receptors) and intrinsic (mitochondria) pathways. Caspase-8 cleaves Bid, which releases cytochrome c, bridging the extrinsic and intrinsic pathways. Cleavage of Bid may amplify caspase-8-induced neuronal cell death following seizures. In the present study, we explored the effect of an inhibitor of caspase-8 (z-IETD-fmk) on the release of Smac/DIABLO and cytochrome c from mitochondria. Rats received intra-amygdaloid injection of kainic acid (KA) to induce seizures for 1 h. The seizures were then terminated by diazepam (30 mg/kg). The damaged and surviving neurons in hippocampus were observed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of caspase-8, Bid, XIAP, caspase-9, cytochrome c and Smac/DIABLO were detected with immunofluorescence and Western blot. The cleavage of caspase-8 and Bid increased at 0 h, cytosolic fraction of cytochrome c and Smac/DIABLO increased by 2 h, cleavage of caspase-9 was detected by 4 h, TUNEL-positive neurons appeared at 8 h and reached a maximum at 24 h following administration of diazepam in the ipsilateral CA3 subfield of hippocampus. Inhibition of caspase-8 significantly decreased neuronal cell death, accompanied by reduction of t-Bid, cleaved caspase-9 and cytosol cytochrome c. Smac/DIABLO from mitochondria was not affected. These results suggest that seizures can lead the translocation of cytochrome c into the cytosol, and the activation of caspase-8 occurs upstream the mitochondria release of cytochrome c and Smac/DIABLO. Inhibition of caspase-8 attenuated neuronal cell death following seizures by decreasing mitochondria release of cytochrome c but not Smac/DIABLO.

Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil, a Rho kinase inhibitor

The migration of aberrant inflammatory cells into the central nervous system plays an important role in the pathogenesis of demyelinating diseases potentially through the Rho/Rho‐kinase (Rock) pathway, but direct evidence from human and animal models remains inadequate. Here we further confirm that Fasudil, a selective Rock inhibitor, has therapeutic potential in a mouse model of myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE). The results show that Fasudil decreased the development of EAE in C57BL/6 mice. Immunohistochemistry disclosed that expression of Rock‐II in the perivascular spaces and vascular endothelial cells of spleens, spinal cords, and brains was elevated in EAE and was inhibited in the Fasudil‐treated group. T‐cell proliferation specific to MOG35–55 was markedly reduced, together with a significant down‐regulation of interleukin (IL)‐17, IL‐6, and MCP‐1. In contrast, secretion of IL‐4 was increased, and IL‐10 was slightly elevated. There were no differences in the percentages of CD4+CD25+, CD8+CD28−, and CD8+CD122+ in mononuclear cells. Histological staining disclosed a marked decrease of inflammatory cells in spinal cord and brain of Fasudil‐treated mice. These results, together with previous studies showing the inhibitory effect of Fasudil on T‐cell migration, might expand its clinical application as a new therapy for multiple sclerosis by decreasing cell migration and regulating immune balance.