brain of these mice [16,17]. These observations suggest that Que may address two key aspects involved in the pathophysiological process of MS, namely prevention of demyelination and modu- lation of the local glial activation, which suggests broader potentials for Que in demyelination diseases. However, it is still unknown whether Que displays an immune-modulating role and may be a promising candidate for treatment of MS. To investigate the potential efficacy of Que as a therapeutic agent, we utilized the MOG induced EAE mouse model to mimic MS. We demonstrate that Que dramatically attenuates the severity of EAE symptoms, diminishes demyelination and the infiltration of CD4+/CD8+ T cells, as well as activation of local microglia in the spinal cord. Additionally, our results indicate that Que exerts immunomodulatory capacities to attenuate MOG35– 55-specific immune response, and to inhibit effector T cell proliferation and thus reduce peripheral CD4+/CD8+ T popula- tion as administrated to EAE mice. Overall, our findings demonstrate the utility of Que as a potential therapeutic agent for demyelinating diseases such as MS. Materials and Methods EAE Mice Model and Treatment Protocols C57BL/6 mice were obtained from the Animal Center of Third Military Medical University. All experiments were performed in accordance with Health Guide for the Care and Use of Laboratory Animals, with the approval of Third Military Medical University Committee on Animal Care (permission NO: SCXK- JUN-2007-015). Female C57BL/6 mice (N = 43, 8 weeks old) were immunized subcutaneously with 200 mg of MOG35–55 peptide (Invitrogen, Carlsbad, CA) emulsified in complete Freund adjuvant (CFA, Sigma Aldrich, Saint Louris, MO) on Day 0 and Day 7, and received 300 ng pertussis toxin (PT, List Biological Laboratories, Campbell, CA) in 0.1 ml PBS intraperitoneal at the time of immunization and 48 hours later. The control (N = 10) mice were immunized with bovine serum albumin (BSA) at the Que Inhibits T Cell Proliferation same dosage (200 mg) followed by PT and the unimmunized mice (N = 9) were given only PBS and PT. Onset and clinical scores of EAE symptoms were evaluated daily using a neurological score as follows: 0, no clinical signs; 0.5, partially limp tail; 1, paralyzed tail; 2, loss in coordinated movement; hind limb paresis; 2.5, one hind limb paralyzed; 3, both hind limbs paralyzed; 3.5, hind limbs paralyzed with weakness in forelimbs; 4, forelimbs paralyzed; 5, moribund as previously described [18]. Quetiapine Treatment Protocols Quetiapine (AstraZeneca, Wilmington, DE) (dissolved in distilled water) was orally administrated to the mice (10 mg/kg/ day) [14]. Que was administrated orally in EAE groups (Que treated) on Day 16 after immunization and maintained for 24 days to test clinical symptoms (40 days after immunization) and histology changes were detected on 30 days after immunization, a time at which apparent histopathology changes can be detected, while those in the untreated groups were given only distilled water. To test immunoresponse in periphery immune organs, Que was orally administrated 1 week before immunization, and mice were sacrificed on Day 10 after immunization, a time at which very efficient MOG35–55-specific responses can be detected. Immunocytochemical Staining and Quantification On Day 30 after immunization, mice were deeply anesthetized with 1% pentobarbital and transcardially perfused with 4% paraformaldehyde in PBS. Spinal cords were dehydrated in 30% sucrose and crossly cut (20 mm) using a cryostate microtome (MS 1900, Leica). Sections were blocked with 10% BSA and incubated with rat anti-CD4, CD8, CD68, CD11b antibodies, or goat anti-MBP, rabbit anti-NG2, mouse anti-APC, and mouse anti-GFAP antibodies (Table S1) overnight at 4uC followed by using an Alex Fluor 488, 568 or Cy5-conjugated secondary antibody relatively. The results were examined under a fluores- cence microscope (90 i, Nikon,) or a laser confocal scanning microscope (PV100, Olympus) with the excitation wavelengths Figure 1. Post-treatment of Que rescues EAE mice from deterioration. A: Schematic diagram displaying the time course of immunization and Que post-treatment. B: Que post-treatment was initiated on day 16 post-immunization when mice attained a clinical score of 0.5 (arrow). Untreated mice (N = 9) continued to deteriorate with increasing clinical scores that reached values of approximately 3. Mice treated with Que (N = 9) with an initial score of 0.5 reached a value just greater than1, and were stabilized and maintained at that value. Values shown are means 6 SEM (*, p,0.05). doi:10.1371/journal.pone.0042746.g001 PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42746