The Tel Aviv University Animal Welfare Committee approved all procedures.
The transgenic mice used in this study (kindly provided by Professor David Ginsburg, University of Michigan, Ann Arbor, MI, USA) carry the ortholog of the human FVL mutation previously generated by a knock-in of the R504Q mutation into the endogenous murine factor V locus by homologous recombination . These mice were back-crossed to C57BL/6 mice for more than seven generations. Genotyping of the offspring for the FVL transgene was performed by PCR with previously described primers, using DNA obtained from tail biopsies taken post-weaning. The mice were raised under standard conditions, 23 ± 1°C, 12 -hour light cycle (0700 to 1900 hours) with ad libitum access to food and water.
Preparation of β2-GPI
Human plasma was used as a source of β2-GPI by the method of Polz et al. . In brief, serum proteins were precipitated by perchloric acid, and the remaining supernatant was adjusted to pH 8 by adding a saturated Na2CO3 solution. This fraction was dialysed exhaustively against 0.03 M NaCI pH 8 at 4°C, and further purified by affinity chromatography on heparin column (HiTrap Heparin HP, GE Healthcare Life Sciences, UK). Fractions containing β2-GPI were eluted with 0.35 mol/l NaCl, then separated by protein electrophoresis and visualized with silver stain. Fractions used for immunization contained a major band that was shown by western blotting to cross-react with a commercial antibody to β2-GPI (anti-ApoH; CSL Behring, Marburg, Germany) .
Induction of experimental antiphospholipid syndrome
Mice heterozygous (FVLQ/+) and homozygous (FVLQ/Q) for the FVL transgene were immunized by a single intradermal injection with 10 μg of β2-GPI emulsified in complete Freund’s adjuvant (CFA). The control group comprised FVLQ/+ mice immunized similarly with CFA. C57BL/6 mice were immunized with either β2-GPI in CFA or CFA alone.
In the first experiment, both female and male FVLQ/+ mice were divided into two groups of fifteen each. Each group included seven to eight mice immunized with β2-GPI (eAPS mice), and seven to eight mice immunized with CFA (adjuvant-immunized controls). In the second experiment, female FVLQ/Q mice (n = 7) were immunized with β2-GPI, and female FVLQ/+ mice (n = 8) were immunized with CFA. Mice were immunized at 3 to 4 months of age, and behavioral assessment was started 4 months later with the staircase test, followed by the elevated plus-maze test and the swim T-maze test on the following sequential days.
For serological evaluation, blood samples were collected from all the mice described above at 1 and 5 months after immunization. Autoantibody measurements were additionally performed in naive FVLQ/+ mice (n = 7), and naive C57BL/6 mice (n = 9). Autoantibody levels in these experiments were also compared with those in C57BL/6 mice with experimental APS induction (n = 10 and n = 11 for C57/B6-APS and C57/B6-control mice, respectively).
Blood samples were collected by retro-orbital sinus puncture as soon as the mice completed their behavioral and cognitive assessment. The sera were separated by centrifugation and stored at −70°C until assayed. The sera were tested by standard ELISA for the presence of autoantibodies as previously described , using serum-dependent (β2-GPI) and serum-independent antibodies to cardiolipin (CL) and phosphatidylserine, and antibodies to β2-GPI and double-stranded DNA.
The staircase apparatus consisted of a polyvinylchloride (PVC) enclosure with five identical steps, 75 × 100 × 25 mm, on top of each other. The inner height of the walls above the level of the stairs was consistent (125 mm) along the whole length of the staircase. The box was placed in a room with constant lighting and isolated from external noise. Each mouse was tested individually. The animal was placed on the floor of the staircase with its back to the staircase. The number of stairs climbed and the number of rears during a 3-minute period were recorded. Climbing was defined as each stair on which the mouse placed all four paws; rearing was defined as each instance the mouse rose on hind legs (to sniff the air), either on a stair or leaning against the wall. The number of stairs descended was not taken into account. Before each test, the box was cleaned with a diluted alcohol solution to eliminate smells.
A three-arm, walled T-maze,constructed of white Plexiglas (600 mm along the stem, 800 mm side at the T-intersection, 400 mm high, with passages 100 mm wide), was situated in one corner of a brightly lit behavioral-testing room separate from the colony. The T-maze was refilled daily with 145 mm of water at 2°C so that a platform (140 mm high, 300 mm2 in size), rising from the floor of the maze, was submerged 5 mm below the water line. One day prior to initial training, mice were placed in the maze and allowed to swim for 60 seconds with no platform present. The platform was then inserted in a standardized position 80 mm from the end of a goal arm, and each mouse was placed directly on the platform for 30 seconds. Finally, each mouse was placed at the far end of the stem and allowed to locate the submerged goal-arm platform. On each of four consecutive training days, a forced-choice alternation paradigm required each subject to perform eight replications of a paired forced-choice/free-choice trial sequence. With either the left or right goal arm blocked with a guillotine door, each subject was placed in the far end of the stem, and allowed to ascend the submerged platform located in the goal arm opposite the blocked arm. The animal remained atop the platform for 15 seconds at the conclusion of this forced choice trial. The animal was then removed by the tail and again placed at the end of the stem, while simultaneously, the guillotine door was removed and the platform moved to the opposite goal arm; that is, the one previously blocked. The latency period for the mouse to reach the platform and the number of correct choices, defined as entry into the goal arm with a platform prior to entry into the goal arm without a platform or re-entry into the stem, were measured during these free-choice trials. Each subject was again allowed to remain on the platform for 15 seconds and was then replaced in the home cage. The goal arm designated for forced choice (right or left hand) alternated from trial to trial over the eight trials of a daily session, from animal to animal over the course of a single day’s testing, and from day to day in terms of trial 1. Subjects who failed to locate the platform within 1 minute were assigned a latency of 60 seconds, lifted from the water by the tail, and placed atop the platform. The results were analyzed as the percentage of correct choices, using repeated-measures ANOVA.
Elevated plus-maze test
The elevated plus maze was made from polyvinylchloride, and built in the shape of a plus sign, with two open (white) arms (340 × 75 × 10 mm) and two closed (black) arms (340 × 75 × 175 mm) opposite each other. The center of the four arms comprised the middle square (75 × 75 mm). The maze was elevated 510 mm above ground level. Each mouse was placed separately in the centre of the maze, facing an open arm, and allowed to explore the apparatus freely for 5 minutes. Parameters measured included the number of entries into the closed and open arms (an index of motor function), and the length of time spent in the closed and open arms. An entry was counted only after the mouse entered the arm with four paws. Before each test, the box was cleaned with a diluted alcohol solution to eliminate smells. The percentage of entries into the open arms out of the total number of arm entries and the percentage of time spent in the open arms, which are all accepted measures of anxiety levels, were further calculated.
Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg) and underwent transcardiac perfusion with phosphate buffer saline followed by perfusion with 4% paraformaldehyde in PBS. Brain tissue was collected, fixed in 4% paraformaldehyde and embedded in paraffin wax. Coronal sections 6 μm thick were cut, mounted, and stained with hematoxylin and eosin (H&E), Luxol Fast Blue (LFB), and Bielchowsky (BLS) stains to identify histological details and the density of myelin and axons, and the sections were specifically examined to evaluate ischemic pathology such as micro-infarcts.
Paraffin wax-embedded sections were dewaxed and rehydrated in xylene and alcohol solutions, then rinsed with PBS. Citrate buffer was used for antigen retrieval, and endogenous peroxidase was blocked with 3% H2O2 in methanol. After incubation of the sections in blocking buffer (Foetal bovine serum, FBS) they were treated with primary antibodies against glial acidic fibrillary protein (GFAP; Dako, Glostrup, Denmark), MAC3, B220 (both BD Biosciences, Inc., San Jose, CA, USA), CD3 (Neomarkers Inc., Fremont, CA, USA), vascular endothelial growth factor (VEGF; Spring Bioscience Corp., Pleasanton, CA, USA), for the detection of astrocytes, macrophage/microglia, B cells, T cells, and VEGF, respectively (dilutions: 1;500, 1:100, 1:100, 1:150, 1:100, respectively). Immunoreactivity was visualized with a commercial system (EnVision HRP; Dako) and sheep anti–rat antibody (AbD Serotec, Raleigh, NC, USA). DAB (Sigma Chemical Co., St Louis, MO, USA) was used as chromogen. Counterstaining was performed with hematoxylin.
Immunofluorescence staining for Iba1 (rabbit polyclonal antibody, Wako, Osaka, Japan) was used to identify microglial and macrophage populations, and was performed using the same protocol as described above with the appropriate secondary antibody (goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (AlexaFluor 488). Slides were mounted with DAPI (Invitrogen Corp., Carlsbad, CA, USA).
In vitro immunohistological staining
We investigated which specific brain structures the aPL antibodies bind to, using brain sections of normal mice immunostained with pooled serum from FVL-eAPS and control mice. Normal brain sections were stained with pooled serum (diluted 1:200) overnight at 4°C and then with the secondary antibody (alkaline phosphatase conjugated anti-mouse IgG). Bound antibody was detected by development with Fast Red substrate (Sigma Chemical Co.) for 10 minutes, after which the sections were mounted with glycerol.
Sections were examined using fluorescence and optical microscopy (Axioplan-2; Carl Zeiss, Jena, German) with the aid of a CCD camera (DS-5Mc; Nikon, Tokyo, Japan) by two independent observers blinded to the experimental groups. The evaluation was performed for the whole brain, using the Paxinos and Franklin (2004) stereotaxic coordinates (ranging from bregma 2.22 to bregma −6.36) . On average, 20 optical fields per slice and three slides per each group were examined under × 20 or × 40 magnification. Measurements were performed with ImageJ software (version 1.43; http://rsb.info.nih.gov/ij/), and data are presented as positive cells per mm2 for MAC3, CD3, B22O, VEGF, and GFAP. Additional evaluation for astrocytic and microglial activation was performed as the ratio of tissue area positive for GFAP/Iba1 per mm2 of total area studied. The density of myelin and axons was evaluated with Image J software, using a range of 0.05 to 3.05 OD units, and measurements were performed using a Rodbard function.
Levels of antibodies and scores on the staircase and plus-maze tests were compared using one-way ANOVA followed by least squares difference post hoc tests. Performance on the swim T-maze was analyzed by means of repeated-measures ANOVA. Most statistical tests were performed using the SPSS software package for PC (SPSS Inc., Chicago, IL, USA). Statistical analysis of histological data was performed using GraphPad Prism software (version 5.0, GraphPad Software, La Jolla, CA, USA). The normality was tested using the Shapiro-Wilk and Kolmogorov-Smirnov tests. Non-parametric data were analyzed using the equivalent Kruskal-Wallis test followed by Dunn’s post hoc multiple-comparison test. Values of all scale data are expressed as mean ± SE. All determinations were made with 95% confidence interval and were considered significant at P < 0.05.