Animals, EAE induction, and disease follow-up
Dark agouti (DA) (Harlan, Italy) female rats, 151–167-g body weight, were used in the first part of the study, placed on ad libitum food and water, and housed three per cage on a 12-h light/dark cycle. The DA rat model of EAE mimics certain aspects of the clinical course of the disease in people with RRMS [17–19], typified by progressive, sustained demyelination, and associated axonal loss [20, 21]. The disease onset in this strain bears is characterized by neurological impairments, manifested as a flaccid tail followed by an acute attack with disturbed gait and paralysis. Most DA animals recover spontaneously from paralysis and experience remission and may then undergo one or more relapses.
A group of 42 rats was sensitized (considering group composition and two extra animals) by a medium containing 0.15 mg/ml guinea pig spinal cord tissue in complete Freund’s adjuvant (CFA, Sigma, Saint Louse, USA), 50% v/v, to which 5 mg/ml of heat-inactivated Mycobacterium tuberculosis (Difco H37Ra, DB, Milan, Italy) was added. Sensitization was performed by injecting 100 μl in both hind pads. Control rats (n = 8) and adjuvant-injected rats (CFA, 50% v/v, heat-inactivated M. tuberculosis, 5 mg/ml) (n = 15) were used. The rats were weighed daily and examined for clinical signs of EAE, according to the following semi quantitative score for neurological disability: 0 = no signs, 1 = loss of tail tone, 2 = mono or bilateral weakness of hind legs or middle ataxia, 3 = ataxia or paralysis, 4 = severe hind legs paralysis, and 5 = severe hind leg paralysis and urinary incontinence [22]. In view of the animals’ disability, wet food was included inside the cages to facilitate feeding. At 1, 5, 8, 11, and 18 DPI, eight EAE rats were randomly sacrificed. From each experimental group, five animals were used for proteomic and molecular biology experiments and three for morphology and immunohistochemistry. All animal protocols described herein were carried out in accordance with the European Community Council Directives (86/609/EEC), approved by the intramural ethical committee for animal experimentation of Bologna University and the Ministry of Health (no. 158/2013-B) and comply with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals [23].
GW2580 treatment and CSF1R inhibition
In order to calculate the number of animals needed to study the effect of the treatment with GW2580, we performed a power analysis using the G*Power 3.1 software. To reach a power of 0.9, based on retrospective analysis of recent research done by others [24, 25], we needed a minimum of n = 6 animals per experimental group.
The experiments were designed in compliance with the ARRIVE guidelines, randomizing the procedures and applying blinded analysis.
GW2580 (LC Laboratories, Boston, USA) was dissolved in 0.5% hydroxypropyl methylcellulose and 0.1% Tween 80 [26, 27]. The experimental groups made of n = 6 animals each were as follows: vehicle (0.5% hydroxypropyl methylcellulose/0.1% Tween 80); control + GW2580; EAE; and EAE + GW2580. The rats (control + GW2580 and EAE + GW2580 groups) were under GW2580 treatment at 40 mg/kg once daily by oral gavage using flexible plastic feeding tubes FTP-15-78-50 (Instech Laboratories, Netherlands) for 1 day prior and 11 consecutive days following the immunization. All animals were weighed daily and the EAE and EAE + GW2580 groups were examined for clinical signs of EAE till the 18 DPI (last day of the experiment) following the semi quantitative score for neurological disability as already explained in the first part of the study. Animal protocols described herein were carried out in accordance with the European Community Council Directives (86/609/EEC), approved by the intramural ethical committee for animal experimentation of Bologna University and the Ministry of Health (no. 607/2016-PR).
Histology and immunohistochemistry
On post-immunization days 1, 5, 8, 11, and 18, three rats were scarified and the lumbar spinal cord (LSC) tissues of the control, EAE, and adjuvant groups were fixed in 4% paraformaldehyde and picric acid and saturated aqueous solution in 0.1 M Sörensen buffer pH 7 for 24 h and then washed with 5% sucrose solution every day till the fixative was completely removed. The tissues were frozen with CO2 gas and kept at −80 °C until processed. Coronal sections (14-μm thickness) were then prepared (Microm HN550, Bio-Optica, Milan, Italy) and processed for morphological studies. For immunohistochemistry, the sections were first rehydrated and then incubated for 1 h with PBS–0.3% Triton X-100, 2% normal serum goat, and 1% BSA, followed by incubation with the primary antibodies diluted in the pre-absorption solution overnight at 4°C. The primary antibodies and dilutions used were as follows: rabbit anti-CD44 (1:100, Cluster of differentiation 44, Acris Antibodies, Inc, San Diego, USA), rabbit anti-CD163 (1:100, Cluster of differentiation 163, Bioss Inc, Woburn, USA), mouse anti-CD86 (1:250, Cluster of differentiation 86, Novus Biological Europe, Cambridge, UK), mouse anti-NF200 (1:200, Neurofilament, Sigma, Saint Louse, USA), rabbit anti CSF1 (1:100, colony-stimulating factor 1, Novus Biological), mouse anti-OX42 (1:250, AbD Serotec Inc., Raleigh, USA), mouse anti-NG2 (1:100, membrane-spanning chondroitin sulfate proteoglycan, Millipore, Merck S.p.a., Milan, Italy), mouse anti-CNPase (1:250, 2′, 3′-cyclic nucleotide 3′-phosphodiesterase, Millipore, Merck S.p.a., Milan, Italy), rabbit anti-GFAP (1:500, glial fibrillary acidic protein, Dako, Milan, Italy), and mouse anti-GFAP (1:500, Sigma, Saint Louse, USA). FluoroMyelin™ Fluorescence Myelin Staining (Molecular Probes, Eugene, OR) was also performed to stain the myelin sheaths, following the manufacturer’s specifications. After rinsing in PBS, the sections were incubated at 37 °C for 30 min with the secondary antibodies: DyLight488-conjugated affinity-pure goat anti-mouse IgG (ThermoScientific, Milano, Italy), Rhodamine Red™-X-conjugated affinity-pure donkey anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA), DyLight488-conjugated affinity-pure donkey anti-rabbit IgG (ThermoScientific, Milano, Italy), and Red™-X-conjugated affinity-pure donkey anti-goat IgG (Jackson Immunoresearch, West Grove, PA, USA) diluted in PBS–0.3% Triton X-100, 1:100. The sections were then rinsed in PBS and mounted in phenylenediamine solution (0.1% 1,4-phenylenediamine, 50% glycerine, carbonate/bicarbonate buffer pH 8.6, Sigma, Saint Louse, USA). The control slices were incubated with the secondary antibodies only and processed in parallel. Images were captured using a Nikon Eclipse E600 microscope equipped with digital CCD camera Q Imaging Retiga-2000RV (Q Imaging, Surrey, BC, Canada). To analyze the inflammatory infiltration, the sections were stained with toluidine blue and evaluated on three replicate sections per animal, counting the number and severity of cellular infiltrates over each whole coronal section. Cellular infiltrates were scored as follows: 0, none; 1, a few inflammatory cells; 2, organization of perivascular infiltrates; and 3, increasing severity of perivascular cuffing with extension into the adjacent tissue [28]. The inflammation score derives from the sum of infiltration scores in each cellular infiltrate.
Spinal cord mRNA analysis
At post-immunization days 1, 5, 8, 11, and 18, five rats were sacrificed and the total LSC was dissected, snap frozen, and stored at −80 °C until used.
The total RNA was prepared from the spinal cord using QIAzol Reagent, cleaned with RNeasy Mini kit (Qiagen; Milano- Italy), and eluted in RNase Free Water, and purity and concentration were evaluated by spectrophotometry using NanoDrop ND-2000 (ThermoScientific, Milano, Italy). Complementary DNA (cDNA) synthesis was performed using RT2 First Strand kit following the manufacturer’s instructions. In brief, after incubation for 5 min at 42 °C with genomic DNA elimination mix in order to avoid any DNA contamination, a reverse-transcription mix of BC3, P2, RE2, and H2O was used and the transcription was performed in a final volume of 20 μl by heating first at 42 °C for 15 min, then at 95 °C for 5 min. Real-time PCR amplification was performed using the Stratagene Mx3005P multiplex quantitative PCR system (Agilent Technologies). The expression of genes involved in MS progression was carried out using RT2 SYBR Green qPCR Mastermix and the Multiple Sclerosis RT2 Profiler PCR Array (Qiagen). The genes are grouped according to the following: T cell activation/signaling, adaptive immunity, cytokine/chemokine inflammation, demyelination, and cellular stress. The raw data obtained was uploaded into GeneGlobe Data Analysis software (SABiosciences, Qiagen) for analysis. Relative quantification of messenger RNA (mRNA) expression was calculated using the comparative cycle threshold (CT) method and is expressed as Log2 fold change of expression. The fold change (2^(−Delta Delta Ct)) is the normalized gene expression (2^(−Delta Ct)) in the test sample divided by the normalized gene expression (2^(−Delta Ct)) in the control sample.
CSF and plasma biomarker analysis
CSF sampling was adapted from the method of Liu and Duff [29]. Briefly, the rats were anesthetized by isoflurane inhalation (Gas Anesthesia System-21100, Ugo Basile, Varese, Italy) and placed prone on the stereotaxic instrument letting the body of the rat laid down. A sagittal incision of the skin was made below the occiput, and the subcutaneous tissue and neck muscles through the midline were separated and held apart using a microretractor. The dura mater of the cisterna magna was then penetrated by an 8-cm long glass capillary, which had a narrowed tip with an inner diameter of about 0.5 mm so that the CSF flowed into the capillary. After collection, each sample was centrifuged at 2000×g for 10 min at 4 °C, and the supernatant aliquoted and stored at −80 °C for biochemical assays.
Blood was collected from the abdominal aorta in EDTA-K2 Vacuntainer tubes and centrifuged at 3000×g for 10 min at 4 °C, and the plasma was collected, aliquoted, and stored at −80 °C until used.
Proteins known to play key roles in neuroinflammation pathways were selected. For this purpose, Bio-Plex Pro™ Rat Cytokine 24-plex Assay (Bio-Rad; Milano, Italy) was used. The kit included EPO, G-CSF (CSF3), GM-CSF (CSF2), GRO/KC, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-17A, IL-18, M-CSF (CSF1), MCP-1 (CCL2), MIP-1α (CCL3), MIP-3α (CCL20), RANTES (CCL5), TNF-α, and VEGF.
The simultaneous quantification of the different proteins in CSF and plasma was performed using xMAP technology and a MAGPIX Luminex platform. This technology makes use of different populations of color-coded beads of monoclonal antibodies specific to a particular protein, thus allowing simultaneous capture and detection of specific analytes from a sample. All the beads from each set are read off, which further validates the results. Using this process, xMAP Technology allows multiplexing of up to 50 unique bioassays within a single sample, both rapidly and precisely [30, 31]. In brief, after the incubation of a specific monoclonal antibody conjugated bead population with 50 μl of CSF/plasma samples for 1 h at RT, washed beads were incubated with detection antibody solution at RT for 30 min, then with the streptavidin–phycoerythrin conjugated solution (RT, 10 min). After washing, beads were resuspended in the assay buffer, shaken for 1 min and then a reading performed on the MAGPIX instrument. The results were analyzed with xPONENT 4.2 ® software and expressed as pg/ml.
Statistical analysis
Student’s t test to compare means of two experimental groups, one-way ANOVA followed by Dunnett’s multiple comparison tests, and two-way ANOVA followed by Bonferroni post-test were used. Data are presented as mean ± standard error of the mean, and significance was set at P ≤ 0.05. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software).
For the normalization of gene expression on the RT2 PCR Profiler Array, five housekeeping genes, ribosomal protein, large, P1, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13A, lactate dehydrogenase A, and actin beta were used. The CT was determined for each sample and normalized to the average CT of the five housekeeping genes. A comparative CT method was used to calculate relative gene expression. Data are represented as Log2 fold change relative to control. The P values were calculated on the basis of a Student’s t test of the replicate 2^(–Delta Ct) values for each gene in the control group and treatment groups, and P values less than 0.05 was considered significant.