Patient samples and IgG purification
Sera/plasma were obtained from 13 NMOSD patients who were AQP4-IgG-seropositive as detected by cell-based indirect immunofluorescence assay [19] and 3 healthy subjects. IgG from sera or plasma was isolated using HiTrap Protein G Sepharose columns (GE Healthcare Bio-sciences, USA). Samples were further purified with Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, USA) and concentrated with Amicon Ultra-15 centrifugal filters (Merck Millipore, Germany). Protein concentration was measured by Bradford assay (Bio-Rad, USA). Pooled IgG isolated from AQP4-IgG-seropositive NMOSD patients was termed IgG(AQP4+). Pooled IgG isolated from healthy subjects was termed IgG(con).
Mice
Female wild-type C57BL/6 mice of age 6-8 weeks were used. Mice were housed in the animal facilities at the Laboratory Animal Unit of The University of Hong Kong. They were maintained in groups of five per cage under a 12 h dark/light cycle and provided with free access of water and chow.
Disruption of BBB and passive transfer of IgG from NMOSD patients
Animal procedures are summarized in Fig 1a. To disrupt the BBB, mice were anaesthetized with intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). They received subcutaneous injections of complete Freund’s adjuvant (CFA, BD Biosciences, USA) containing heat-killed H37Ra Mycobacterium tuberculosis (Difco, USA) at 4 sites (50 µg in 50 µl CFA each site) on the hind flank on 7 days before IgG transfer. In addition, mice received i.p. injections of pertussis toxin (PTx, 200 ng in 0.2 ml PBS, List Biological Laboratories, USA) on 7 and 3 days prior to IgG transfer.
Drug treatments
Memantine (Sigma-Aldrich, USA) was prepared by adding the compound to corn oil (Sigma-Aldrich, USA). The mixture was sonicated at room temperature for 20 min. It was freshly made immediately before each administration. Following BBB disruption, mice were randomly assigned to four sets of experiments. For preventive treatment, IgG(AQP4+) mice in set 1 received i.p. injection of 4 mg purified IgG from AQP4-IgG-seropositive NMOSD patients and oral gavage of vehicle or 60 mg/kg body weight memantine on the day of IgG passive transfer (day 0). Animals were sacrificed at 1 day post-injection (dpi). In set 2, beginning from day 0, IgG(AQP4+) mice received daily i.p. injection of purified IgG from AQP4-IgG-seropositive NMOSD patients and oral gavage of vehicle or memantine till 3 dpi. Animals were sacrificed at 4 dpi. In set 3, beginning from day 0, IgG(AQP4+) mice received daily i.p. injection of purified IgG from AQP4-IgG-seropositive NMOSD patients and oral gavage of vehicle or memantine till 7 dpi. Animals were sacrificed at 8 dpi. For therapeutic treatment in set 4, IgG(AQP4+) mice received daily i.p. injection of purified IgG from AQP4-IgG-seropositive NMOSD patients beginning from day 0 till 7 dpi. Oral gavage of vehicle or memantine was begun on the 4 dpi and continued once daily immediately after IgG injection till 7 dpi. Animals were sacrificed at 8 dpi. IgG(con) mice which received vehicle were used as sham controls in all sets of experiments. The dose of memantine was adopted from a previous study [16].
Detection of motor impairments
Motor impairments were detected using beam walking test, in which examines the animal’s ability to keep upright and walk across an elevated narrow beam to a platform [20]. The apparatus consisted of 80 cm long beams with width of 1.2 or 0.6 cm, resting 50 cm above a table top on two stands. Prior to CFA and PTx treatment, mice were trained for 2 days, with 3 trials a day, with walking across each of the 12 and 6 mm-wide beams. One day after completion of IgG transfer and drug treatment, the time for the mice to cross each beam and the number of paw slips during the process were recorded.
Tissue preparation and immunofluorescence
After beam walking test, mice were sacrificed by pentobarbital overdose and received intracardiac perfusion with ice-cold PBS and paraformaldehyde. Cervical spinal cords were harvested, and sectioned at 10-µm thickness with a cryostat (Thermo Fisher Scientific, USA). Immunofluorescence was performed with standard procedure. The following primary antibodies were used: (1) rabbit anti-human IgG (1:600, Dako, USA), (2) rabbit anti-AQP4 (1:200, Sigma-Aldrich, USA), (3) mouse anti-glial fibrillary acidic protein (GFAP, 1:200, Santa Cruz Biotechnology, USA), (4) goat anti-myelin basic protein (MBP, 1:200, Dako, USA), (5) rabbit anti-neurofilament heavy polypeptide (NF-H, 1:400, Sigma-Aldrich, USA) (6) rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1, 1:200, Wako, Japan), and (7) rat anti-lymphocyte antigen 6 complex locus G6D (Ly6G, 1:400, Abcam, UK). Sections were then incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies (Thermo Fisher Scientific, USA) at room temperature for 1 h. They were counterstained with DAPI and mounted with antifade reagent (Thermo Fisher Scientific, USA).
Image processing and quantification
Measurement of immunofluorescent intensity was performed on eight rostral-to-caudal alternate cross sections of the ventrolateral white matter of the cervical spinal cord. All signals were captured with the same microscope (Nikon Eclipse Ni, Japan) and digitized with SPOT software 5.0 (Diagnostic Instruments, USA) in identical settings. Signal intensity was quantified using ImageJ software (Wayne Rasband, NIH, USA).
Histochemistry
Luxol fast blue (LFB) staining was performed according to the manufacturer’s instructions (Abcam, UK). Briefly, spinal cord sections were rehydrated and incubated in 0.1% Luxol fast blue solution at 40oC overnight. Excess stain was rinsed with 95% ethanol. Slides were washed in distilled water, immersed in 0.05% lithium carbonate solution for 30s, and then dehydrated in serial ethanols and mounted in Permount mounting medium (Sigma-Aldrich, USA). In addition, selected sections were stained with hematoxylin and eosin (H&E) using standard procedure.
Detection of apoptotic cells
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using the In-situ Cell Death Detection Kit (Roche, Germany) according to the manufacturer’s instructions. Sections were mounted by counterstained with DAPI and mounted with antifade reagent (Thermo Fisher Scientific, USA). To quantify apoptotic cells, cells positive for TUNEL with condensed nuclei were counted on eight rostral-to-caudal alternate cross sections of the ventrolateral white matter of the cervical spinal cord.
ELISA for cytokines and growth factors
Following transcardiac perfusion with ice-cold PBS, spinal cords of mice were harvested and homogenized using a lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor. Levels of proinflammatory cytokines including interleukin-1β (IL-1β; RayBiotech, USA), interleukin-6 (IL-6; RayBiotech, USA) and tumor necrosis factor-α (TNF-α; RayBiotech, USA), and growth factors including brain-derived neurotrophic factor (BDNF; MyBioSource, USA), glial cell line-derived neurotrophic factor (GDNF; MyBioSource, USA) and vascular endothelial growth factor (VEGF; MyBioSource, USA) in the homogenates were determined using mouse ELISA kits according to the manufacturer’s instructions.
Statistical analysis
Differences between groups were compared by one-way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test. Data are shown as mean ± SEM. Levels of significance are indicated with *p < 0.05, **p < 0.01, and ***p < 0.001. Calculation was performed using IBM SPSS Statistics Version 24 software.