Purified recombinant AQP4-IgG (rAb-53) [3, 5] was provided by Dr. Jeffrey Bennett (Univ. Colorado, Denver) and (non-NMO) pooled human IgG, as control, was purchased from Pierce Biotechnology (Rockford, IL). Some studies were done using an antibody fragment-based complement inhibitor, herein called Compinh (to be reported separately), which inhibits serum complement activity by >95% for 8–12 h after intravenous administration. Unless otherwise specified chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
CD59−/− rats in a Sprague-Dawley background were custom-generated by Transposagen Biopharm. Inc. (Lexingtobon, KY) using CRISPR-Cas9 gene targeting technology as described . In vivo studies were done on 8- to 10-week-old, weight-matched CD59+/+ and CD59−/− rats. Rats were maintained in air-filtered cages and fed normal rat chow in the University of California, San Francisco (UCSF) Animal Care facility. All procedures were approved by the UCSF Committee on Animal Research (approval number AN108551).
Blood and urine analysis
Blood (800 μL) was collected before and 24 h after AQP4-IgG injection. Hematological parameters were measured using a Genesis Hematology Analyzer (Oxford Science, Oxford, CT) in which 80 μL blood was collected into a MiniCollect tube with EDTA (Greiner Bio-One GmbH, Kremsmunster, Austria). Serum was obtained by collection of blood in 1.5-ml Eppendoff tubes then allowing clotting for 30 min followed by centrifugation at 800 g for 10 min at 4 °C. Serum was frozen at −70 °C for blood chemistry analyses (IDEXX BioResearch, Sacramento, CA). Urine was collected for determination of osmolality using a freezing-point depression osmometer (Model 3320 Osmometer, Advanced Instruments Inc., Natick, MA).
Adult CD59+/+ and CD59−/− rats were administered AQP4-IgG (4 mg/kg body weight in PBS) by intraperitoneal injection in a total volume of 500 μL. Blood was collected through the tail vein at 1, 2, 4, 6, 8, 24 and 48 h, left for 30 min at room temperature to allow clotting, and centrifuged for 10 min at 800 g at 4 °C. Serum was diluted 50,000-fold and human IgG concentration was determined using a human IgG ELISA kit (GenWay, San Diego, CA).
Intraperitoneal injection model
AQP4-IgG or (non-NMO) control human IgG (5 mg/kg body weight) was injected intraperitoneally. At 24 h, clinical motor scores were recorded, as adapted from scoring used for neuroinflammation models in rodents : score 0, normal movement; score 1, tail paralysis; score 2, hindlimb paralysis; score 3, hindlimb and frontlimb paresis with breathing difficulty; score 4, complete paralysis with moribund condition. Then rats were deeply anesthetized using ketamine (100 mg/kg) and xylazine (10 mg/kg) and transcardially perfused with 200 mL heparinized PBS and 200 mL of 4% PFA in PBS. Brain, optic nerve, spinal cord (cervical), skeletal muscle, kidney and stomach were removed and post-fixed for 4 h in 4% paraformaldehyde (PFA) and cryoprotected in 20% sucrose. Tissues were embedding in OCT compound and sectioned at 7-μm thickness using a cryostat (CM1900, Leica) for immunofluorescence. For complement inhibition studies, Compinh (50 mg/kg) was injected intravenously just before and 12 h after intraperitoneal AQP4-IgG (or control IgG) administration.
Frozen sections of harvested tissues were immunostained as described . Briefly, sections were incubated in blocking solution (1% BSA containing 0.3% Triton X-100 in PBS) for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against AQP4 (1:200, Santa Cruz Biotechnology), human IgG (1:200, Santa Cruz Biotechnology), C5b-9 (1:100, Hycult Biotech, PA), CD45 (1:50, Cambridge, MA), CD59 and CD55 (1:50, LSBio, Seattle, WA), CD46 (1:50, Abcam, MA), GFAP (1:200; Millipore), ionized calcium binding adaptor molecule 1 (Iba1; 1:400, Wako, Richmond, VA), or myelin basic protein (MBP, 1:100, Santa Cruz Biotechnology), followed by the appropriate species-specific Alexa Fluor-conjugated secondary antibody for 1 h (5 μg/mL each, Invitrogen) at room temperature. Sections were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen, Life Technologies, Eugene, OR) for visualization of immunofluorescence on a Leica fluorescence microscope or Nikon confocal microscope.
Data are presented as mean ± S.E.M. Statistical analysis was performed using Prism 5 GraphPad Software package (San Diego, CA). The normality of the data was established by Bartlett’s test for equal variances and a one-way ANOVA with Newmann-Keuls post-hoc test to compare groups.