Animals and study design. Male Balb/c mice (2–3 months old) weighting 22–26 g, were obtained and housed in clear plastic cages, under controlled temperature and humidity in a 12 h light/dark cycle with free access to water and food. All animals were allowed to acclimate to their new surroundings for 1 week before they undergo any experimental procedures. All protocols were approved by the ethics committee of Tabriz University of Medical Sciences (approval No. IR.TBZMED.VCR.REC.1398.067). All experiments were performed according to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH Publications).
-54 adult male mice were randomly divided into six groups (n=-9): Sham group (laminectomy surgery without compression injury), sSCI (48h), sSCI (2W), and sSCI (1M) groups (severe compression injury at 8th thoracic segment ‘T8’ of the spinal cord and were sacrificed 48 h, 2 weeks, and 1 months after the SCI), sSCI (2M) + IgG group (severe compression injury at T8 and received IgG after the SCI for 2 months), and sSCI (2M) + cis mAb group (severe compression injury at T8 and received cis mAb after the SCI for 2 months).
Laminectomy and calibrated forceps model of spinal cord compression. All procedures were performed under sterile conditions. Mice were anesthetized with 4% isoflurane, and a laminectomy was done at T7-9 to expose the T8 segment of the spinal cord, without any damage to the dura. Pairs of forceps were applied for laterally compressing the spinal cord to the corresponding thickness (0.25 mm) for 15 sec [39, 40]. Sham group received laminectomy and forceps placement around the spinal cord, without a compression. Muscles and skin were stitched after forceps removal followed by administration of saline solution for rehydration (1 ml), buprenorphine (0.05 mg/kg) to alleviate pain and ciprofloxacin (5 mg/kg) to treat/prevent bladder infection, all subcutaneously (SC) two timed a day for 3 days. Animals were monitored in a temperature‐controlled room until recovery and then transferred to their separate cage. Bladders of SCI mice were manually expressed two times a day until the establishing of the urinary reflex.
Locomotor analysis. Motor function was assessed in mice to ensure that an effective SCI or a successful laminectomy was done. Hind-limb function in groups was tested by the Basso mouse scale (BMS) one day after injury in the open-field (OF) [41]. In brief, animals were individually placed in the OF chamber (22.5 × 22.5 cm) and allowed to freely explore for 5 min. Two evaluators independently gave each animal a score of 0 to 9, with a score of 0 indicating a complete defect in locomotor function and a score of 9 indicating no locomotor deficits.
To determine the characteristics of cis P-tau, AT8 P-tau, and AT100 P-tau induction upon SCI, mice in the sSCI (48h) and sSCI (2W) groups were anesthetized with intraperitoneal (IP) injection of ketamine (60 mg/kg) and xylazine (10 mg/kg) 48 h and 2 weeks after the SCI, and the spinal cord tissue samples were collected for immunoblotting and immunofluorescence staining analyses.
Antibody treatment of mice. To evaluate the efficacy of cis mAb in treating SCI, we examined whether cis mAb could affect intracellular P-tau in sSCI (2M) + cis mAb, and sSCI (2M) + IgG groups. Undergoing sSCI, male mice were randomly treated with mouse cis mAb or mouse IgG. Animals received 1 dose of cis mAb/IgG IP pre-treatment (200 µg/per mouse) 3 days before the injury, single IP post-injury treatment (20 µg in 5 µl) 15 min after SCI, then IP post-treatment (200 µg) every 4 days for 2 weeks, followed by 200 µg weekly for the rest of the two month treatment (33).
Transmission electron microscopy (TEM). The ultrastructural assessment was carried out using the TEM method. The brain and spinal cord specimens, from sham and SCI mouse models treated with either control IgG or cis mAb, were cut into pieces of 2 × 2 mm. Briefly, the cells were fixed in glutaraldehyde 2.5%, buffered at 0.1 M phosphate (pH 7.4), osmium tetroxide 1% was used for post-fixation, and finally embedding was performed using resin. Ultrathin sections around 60–90 nanometer were cut and taken on a copper grid and stained with a mixture of uranyl acetate and lead citrate and examined under a ZIESS electron microscope (EM902A), and then viewed by a Leo 906 (Leo, Germany) transmission electron microscope.
Immunoblotting analysis. For immunoblotting, spinal cord and the brain samples were homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP 40, 0.1% SDS, 0.5% Na-deoxycholate, 50 mM NaF) containing proteinase and phosphatase inhibitors and then mixed with the SDS sample buffer and loaded onto a gel after boiling. The proteins were resolved by polyacrylamide gel electrophoresis 12% and transferred to a PVDF membrane. Then the membranes were blocked with 2% milk in TBST (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 h. Next, the membranes were incubated with primary antibodies overnight at 4ºC. Then, the immunoblots were incubated with HRP-conjugated secondary antibody in 2% milk in TBST. The signals were detected using chemiluminescence reagent (Perkin Elmer, San Jose, CA). The membranes were washed 6 times with TBST after each step. Immunoblotting results were quantified with imageJ. The band of interests were normalized against actin.
Immunostaining analysis. Mice were deeply anesthetized and perfused through the left ventricle with 10% neutral buffered formalin. The spinal cord and brain were removed immediately, and post-fixed in 10% neutral buffered formalin overnight. A 1.5 cm segment of spinal cord centered on the injury site and brain were embedded into paraffin, and then cut into 8 µm increments by a microtome.
Sections were dewaxed, and then dehydrated in a serial dilution of ethanol. A 5% ammonium chloride solution (Merck, 101145) were used to quench autofluorescence. The sections were placed in a steamer (0.01 M) Sodium citrate (Sigma-Aldrich, S4641) for 20 minutes for antigen enhancement. Then, the slides were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, T8532) for 15 min, and blocked with 10% anti-goat serum for one hour. Next, the slides were incubated with following primary antibodies overnight: cis P-tau mAb (gift from KP. Lu), AT8 P-tau (MN1020), and AT100 P-tau (MN1060). The samples were incubated with the anti-mouse secondary antibodies (Alexa Fluor 488 or 594) at 37°C for one hour. The nucli were stained with Dapi (Invitrogen, D1306), and the images were visualized by a fluorescent microscope (BX71; Olympus equipped with DP72 digital camera).
Twenty ROIs (The 350×450 µm2 dimension representative view) from the cortices of the mouse brains were randomly selected and quantified for each group. The immunofluorescence intensity were quantified with ImageJ (n = 3/per group).
Open-field locomotion. On days 1, 30, and 60 post-injury, hind-limbs functions and spontaneous locomotor activity were evaluated in the OF test. We used the BMS and the related subscale to score locomotor performance. To determination of spontaneous locomotor activity, as mice freely explore the OF chamber for 5 min, a computer-based video-tracking system (Noldus Ethovision) recorded total traveled distance and speed. The OF test is not only used to measure motor activity, but also to measure other factors such as anxiety. Rodents usually spend more time in contact with the walls of the chamber than in the center area (thigmotaxis) [42, 43]. On day 60 after injury, the mouse freely explored the maze for 5 min, and the tracking software recorded movement. Mice that spend most of their time in the unprotected central area of the maze indicate anxiolytic-like behaviors [42, 43].
Elevated plus-maze (EPM) test. The EPM task is an experiment model to evaluate anxiety/risk-taking behavior; which examines cortical circuits [44–49]. The test is performed on a plus-shaped apparatus which is raised 50 cm above the ground, with two opposite open (aversive, without walls) and two opposite closed (safe, 15 cm high walls) arms (30 × 5 cm), and a central square. A mouse is placed on the central square of the apparatus, facing an open arm, and explore through the maze for 5 min. The number of open arm entries and total time exploring open arms were recorded (Noldus Ethovision). The maze was thoroughly cleaned after each trial to avoid any odorant confounding the test. Mice with normal levels of anxiety/risk-taking behavior enter the open arms less often and spend less time in the open arms. Entering the open arms is being addressed to abnormal risk-taking and anxiety behaviour associated with a cognitive decline [33, 50].
Y-maze spontaneous alternation test. The Y-maze test is used for the evaluation of spatial working memory or to quantify cognitive deficits in rodents. The Y-maze consists of three identical black arms (30 cm long, 6 cm wide with 15 cm high walls) mounted in the shape of the letter ‘Y’ at a 120° angle from each other. Rodents usually show a tendency to explore a new arm rather than exploring the arms previously visited. One arm (arms A–C) was randomly selected as the ‘start’ arm, and the mouse is placed at the end of it and allowed to move through each arm. The number of arm entries (when all four paws enter the arm) was recorded for 10 min. Alternation is consecutive entries into each arm without any repeats. The alternation percentage is calculated by using the following formula: (Number of alternations / Number of arm entries) × 100. A mouse with intact working memory scored significantly ˃ 50% [51–53].
At the end of the second month, and after the behavioral tests, mice were anesthetized by ketamine-xylazine IP injection. For electron microscopy, immunoblotting, and immunofluorescence staining analyses, samples were collected from the brain and spinal cord tissues.
Statistical analysis. All normally distributed data were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s HSD multiple comparisons post hoc test to compare the groups mean differences. All data were analyzed with SPSS software version 22, and were reported as the means ± standard deviation (SD) with P < 0.05 considered significant.