Pristane induced lupus mice as a model for neuropsychiatric lupus (NPSLE)

Animals

Specific pathogen-free BALB/c mice were purchased from Vital River Laboratory (Beijing, China) at the age of 4 weeks. Female mice were used for the experiment at the age of 8 weeks. All animals were reared in standard animal cages under environmentally controlled laboratory conditions (12/12 h light/dark cycle, 22 ± 2 °C, 40–80% humidity) with ad libitum access to food and water. All efforts were made to minimize animal suffering. The animals were maintained and treated in compliance with the policies and procedures detailed in the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health. The Animal Care and Use Committee of China Medical University reviewed and approved the animal experimental protocols and the treatment procedures (No. KT2018060).

Pristane injection

As shown in Fig. 1, at the age of 8 weeks, mice were randomly divided into the following eight groups (n = 12 per group): 4 control groups (1 m, 2 m, 4 m and 8 m) and 4 PIL groups (1 m, 2 m, 4 m and 8 m), which received a single intraperitoneal injection of 0.5 ml phosphate buffer saline (PBS) or pristane (Sigma-Aldrich, St. Louis, MO, USA), respectively. The dose of pristane used in this study was based on a previous research [73]. Mice were sacrificed at month 1, 2, 4 or 8 after a battery of behavioral tests. Blood samples were obtained from the eyeball. Tissue samples of spleen, kidney, joint and brain were harvested for further examination. There was no early euthanasia of animals during the study.

ELISA for brain cytokines and chemokines, and serum cytokine, total IgG and autoantibody detection

Mice were anesthetized and transcardially perfused with 0.1 M PBS (pH 7.5, 4 °C). Brains were harvested and dissected into the left and right hemispheres. One hemisphere was snap-frozen in liquid nitrogen and subsequently made into frozen sections for immunofluorescence staining, while the other hemisphere was homogenized on ice in PBS and centrifuged at 12,000 rpm for 15 min at 4 °C to remove cell debris. Supernatants were collected and stored at − 80 °C until assay. Serum was separated by centrifugation at 5000 rpm for 15 min at 4 °C. Cytokines and chemokines in brain tissues and serum were detected at week 1 or 2, or month 1, 2, 4 or 8 using IL-1β, IFN-α, IFN-β, IL-10, IFN-γ, IL-6, TNF-α, IL-17A, BAFF, CCL2 and CXCL10 ELISA kits (Boster & Biological Technology, Wuhan, China), TWEAK and APRIL DuoSet kits (R&D Systems, Minneapolis, MN, USA) and CCL7 ELISA kit (CUSABIO, Wuhan, China). Autoantibodies (anti-chromatin IgG (Inova Diagnostics, San Diego, CA, USA), anti-dsDNA IgG and anti-Sm IgG (CUSABIO), and anti-nRNP IgG (Alpha Diagnostics, San Antonio, TX)) and total IgG (Boster & Biological Technology) in serum were determined using commercially available kits per manufacturer’s guidelines. Each sample was tested at least three times, and the average value was taken.

Renal function and 24 h proteinuria assessment

The levels of Scr and BUN were detected using Scr and BUN assay kits (Jiancheng, Nanjing, China). Experimental procedures were strictly followed according to the manufacturer’s protocols. Urine samples from mice in a metabolic cage were collected over 24 h. Proteinuria was measured with a BCA kit (Beyotime biotechnology, Shanghai, China). Each sample was tested at least three times and the average value was taken.

Hematoxylin and eosin (H&E) staining

Tissues from the hind limb, brain and kidney were harvested and post-fixed in a solution of 4% paraformaldehyde in PBS overnight at 4 °C. The joint tissues were decalcified with ethylenediamine tetraacetic acid (EDTA, pH 8.0) for 2 weeks, and then embedded in paraffin after dehydration, and sliced into 4 μm sections. The sections were stained with H&E to observe synovial damage and provide an anatomic reference for the glomerulus and brain areas.

Arthritis severity score and synovial inflammation score

For quantified scoring of arthritis severity, we used a previously published scoring system [16], as follows: score scale of 0–3, where 0 = normal, 1 = slight swelling or erythema of the wrist/ankle joint or footpad, 2 = moderate swelling and erythema of the wrist/ankle joint or footpad, and 3 = severe swelling and erythema of the paw. The scores for individual limbs were summed to obtain a total arthritis severity score of 12 per animal. Arthritis severity score was assessed twice by two independent observers. For synovial inflammation, we used a scoring system described previously [74], in which 5 high-power magnification fields (HPF) were scored for the percentage of infiltrative mononuclear inflammatory cells, as follows: 0 = absent, 1 = mild (1–10%), 2 = moderate (11–50%), and 3 = severe (51–100%). The average score of the 5 HPFs was used for analyses.

Spleen index

Spleen index was calculated as the ratio of spleen weight to body weight (mg/g) as reported previously [18].

Behavioral assessments

The mice were subjected to a series of behavioral tests in the following order: olfactory sensitivity test, open field test, elevated zero maze test, novel object test, social novelty preference test, rotarod test, PPI test and forced swim test. Mice were tested during the same lighting and time-of-day conditions. All behavioral chambers were cleaned with 70% ethanol as mice were changed. Researchers were blinded to the experimental groupings.

Olfactory sensitivity test

The paradigm used to assess olfactory sensitivity in this study was similar to that in an earlier report [26]. Following habituation to a new test chamber (45 cm × 24 cm × 20 cm), each mouse was introduced to a 5 cm × 5 cm piece of filter paper scented with 0.25 ml of an odorant for 2 min. Then, the scented filter paper was removed, and the mouse was allowed to rest for 1 min. This procedure was repeated three times. Each mouse was presented with repellents (vinegar and alcohol) and attractants (male feces and female feces) diluted with PBS to the same concentration. Test chamber was covered with a clear piece of plexiglas to limit evaporation and entry of external odorants. Active investigation was defined as directed sniffing within 0.5 cm of the odorant source and the sniffing time was recorded. Sniffing time for each trial was summed to obtain a total value per animal.

Open field test

The open field chamber (40 cm × 28 cm × 40 cm) was made up of black polyvinyl chloride panels with a non-reflective base. The central zone was defined as a 20 cm × 14 cm area. Each mouse was positioned individually in the center zone and allowed to freely explore the arena for 30 min. Total distance travelled (km) and time spent in the center (s) were digitally recorded and analyzed using custom-built programs.

Elevated zero maze test

The elevated zero maze was a ring-shaped apparatus, elevated 50 cm from the floor, and consisted of a circular platform (outer diameter 50 cm, width 10 cm) divided into four quadrants of equal length with two open arms and two closed arms (surrounded by a 20-cm wall from the surface of the maze). The test was conducted as previously described [75]. The test mouse was placed at the open arm and was allowed to conduct a 10-min free exploration. Total distance travelled (m) and percentage of time spent in the open arms were digitally recorded and analyzed by custom-built programs.

Novel object recognition test

The novel object recognition test was used to evaluate recognition memory and was conducted as previously described [76, 77]. During the acclimation phase, mice were allowed to habituate to the apparatus (55 cm × 40 cm × 30 cm) with no objects for 10 min, and then a test phase began 24 h later. On the trial day, two identical cylindrical objects were placed in the opposite side of the apparatus, and mice were allowed to spend 10 min with the objects. One hour later, one of the objects was replaced with a triangular object, and time spent exploring the novel and familiar object were digitally recorded for 10 min. The time spent in close interaction with each object was converted into a discrimination ratio, which was calculated as follows: time spent exploring the novel object/total time spent exploring both objects.

Social novelty preference test

The social novelty preference test was performed as previously described, with minor modification [78, 79]. During the acclimation phase, a test mouse was allowed to habituate to the apparatus for 10 min. A stranger mouse was then placed in one of the wire cages. The test mouse was allowed to spend 10 min to explore the entire apparatus. Subsequently, a novel stranger mouse was placed in the other wire cage. The test mouse was allowed to freely investigate the entire apparatus (the familiar mouse in one corner and the novel stranger mouse in the opposite corner) for 10 min. The time spent in close interaction with each mouse was digitally recorded and converted into a discrimination ratio, which was calculated as follows: time spent exploring the stranger mouse/total time spent exploring both mice.

Rotarod test

The rotarod test was used to evaluate motor coordination and was performed as previously described, with minor modification [18]. First, mice were placed on the stationary bar to habituate to the apparatus for 2 min. Then, the rotarod began to accelerate from 4 to 40 rpm. Latency to fall off the rotating rod was recorded with a 5-min cutoff time for three trials per day over 3 consecutive days and the mean retention time on the rod per trial was recorded.

PPI test

PPI test was measured using a startle chamber and was conducted as previously described, with minor modification [80, 81]. The test mouse was given a 10-min acclimation period in the startle chamber during which a 70 dB background noise was presented, and then the test mouse was subjected to test trials consisting of four trial types; that is, one type of startle stimulus only trial and three types of PPI trials. White noise of 120 dB (40 ms) was used as the startle stimulus for all trial types. The peak startle amplitude was recorded with the onset of the startle sound. The prepulse stimulus was presented 100 ms before the onset of the startle stimulus with an intensity of 75, 85 or 95 dB (20 ms). Six blocks of the four trial types were presented in a pseudorandom order such that each trial type was presented once within a block. The intertrial interval had an average duration of 15 s. PPI responses were calculated as follows: PPI% = [1 − (prepulse trials/startle only trials)] × 100.

Forced swim test

Each mouse was placed into a glass beaker containing 3000 ml of water maintained at approximately 24 ± 1 °C. Following habituation to swimming in this glass beaker for 2 min, a 4-min test session was digitally recorded. Mice placed in this situation had no way to escape, and began struggling and swimming, and eventually exhibited behavioral despair, assessed as immobility [34]. Depression-like behavior was defined assessed as the time spent immobile.

Measurement of BBB permeability

To evaluate alterations in BBB permeability, Evans blue dye was used as a marker of BBB leakage, as previously described [82]. Briefly, mice were administered 2% Evans blue dye solution (4 ml/kg, Beyotime biotechnology) intravenously 30 min before sacrifice. Then, the brain tissues were homogenized in 50% trichloroacetic acid at a 1:3 v/v ratio. BBB permeability was assessed as Evans blue extravasation, and was quantified in the supernatant from each sample following addition of 90 μl of 95% ethanol (absorbance, 620 nm).

Immunofluorescence staining

Kidney and brain tissues were dissected, fixed in 4% paraformaldehyde for 24 h, followed by immersion in 30% sucrose (w/v) solution at 4 °C overnight. Tissues were then cut into 10-μm-thick coronal frozen sections, and blocked with 10% goat serum for 30 min at room temperature. Sections were incubated with primary antibodies, including anti-C3 (1:100; Santa Cruz, CA, USA), anti-Iba-1 (1:200; Abcam, Cambridge, UK), anti-GFAP (1:500; Abcam) or anti-NeuN (1:500; Abcam) at 4 °C overnight. On the following day, sections were incubated with secondary antibodies, including Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200; Proteintech, Wuhan, China) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Proteintech, Wuhan, CHN) for 2 h at room temperature in the dark. For locating cell nuclei, sections were stained with DAPI (Beyotime biotechnology) for an additional 8 min. TUNEL staining was performed using a TUNEL Bright Green Apoptosis Detection kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Images were captured on a microscope (BX53, Olympus, JPN) at × 200 or × 400 magnification. The number of stained cells was automatically counted in a defined area using Image J software. The MFI of C3 deposition in the glomeruli was calculated using Image J software. The data from three random sections for each individual mouse were averaged to obtain a single value. For IgG staining, sections were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200, Proteintech) for 2 h at room temperature in the dark. The MFI of IgG deposition in the choroid plexus, lateral ventricular wall or glomeruli was calculated using Image J software. To examine autofluorescent lipofuscin, regions of interest were captured at 480 nm and 550 nm exciting light. The mean gray value of autofluorescent lipofuscin was quantified with image J software.

Statistical analysis

GraphPad Prism V8 software (La Jolla, CA, USA) and SPSS 22.0 software (SPSS Inc., Chicago, USA) were used for statistical analysis. Before applying parametric statistics, all data were checked for the assumptions of normality using the D’Agostino-Pearson omnibus normality test. All data were expressed as the mean ± SEM. Differences in normally distributed data were detected by repeated measures analysis of variance (ANOVA) (with time as within factor and group [PBS, pristane] as between factor) followed by Tukey's post hoc test for multiple comparison. For non-parametric data, the Scheirer–Ray–Hare extension of the Kruskal–Wallis test [83] was used as a non-parametric equivalent of the two-way ANOVA. p < 0.05 was considered a statistically significant difference in all sampled groups.

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