In vitro experiments
Cell Culture and Sphingosine-1-phosphate receptor 5 agonist treatment
Schwann cells of the IFRS1 lineage were obtained from COSMO BIO Co., Ltd. (catalog number: PMC-SWN-IFRS1C) and cultured in a specialized medium specifically designed for this line, purchased from COSMO BIO Co., Ltd. (catalog number: PMC-SWN-IFRS1C-COS). This medium was formulated to provide optimal support for the growth and maintenance of the IFRS1 lineage of Schwann cells, ensuring proper cultivation and suitability for experimental purposes. The cells were plated in 100 mm plastic culture dishes, and incubated at 37°C in a humidified environment with 5% CO2. The cultures were allowed to reach a subconfluent state over a 25-day period.
On day 25, the cells were treated with various concentrations of the S1Pr5 agonist (0, 25, 50, 100, or 200 μM). The S1Pr5 agonist dissolved in dimethyl sulfoxide (DMSO), which also served as a vehicle control. The experimental setup included six conditions: four agonist concentrations and two control groups. One control group was administered a high concentration of DMSO, whereas the other remained untreated.
The messenger RNA Analysis
Twenty-four hours post-treatment, cells underwent RNA harvesting for the purpose of quantifying the mRNA expression of C-Jun and Shh through qRT-PCR. The RNA was extracted using Qiagen's RNeasy® Universal Mini kit, located in Tokyo, Japan, and cDNA was subsequently synthesized utilizing iScript RT SuperMix for qRT-PCR from Bio-Rad, also based in Tokyo, Japan. This cDNA was then employed as a template in real-time PCR, conducted using iTaq™ Universal SYBR® Green Supermix from Bio-Rad, with gene-specific primers obtained from TAKARA BIO (Otsu, Japan) and Bio-Rad. Primers for sequencing are detailed in Supplementary data (Table 1). Semi-quantitative analyses of the mRNA levels of transcription factors associated with Schwann cell regeneration were conducted, referencing an endogenous control on the CFX96 Touch™ Real-Time PCR Detection System from BIO-RAD. These gene expression levels were expressed as relative copy numbers, calculated via the ΔΔCq method, offering a comparative assessment of mRNA levels.
In vivo experiment
Subjects
The 30 female Lewis rats(Charles River Japan, Yokohama, Japan) used in this study were 7 weeks old at the start of the experiment. The rats were housed in the Toho University Ohashi Experimental Animal Laboratory. The gages were maintained at 25 °C with a 12-h light–dark cycle. The rats were provided with a comfortable and clean-living environment, which included proper bedding and enrichment to promote natural behaviors. The diet and hydration were carefully monitored to ensure adequate nutrition. Regular health checks were conducted to promptly identify and address any health issues. Additionally, all procedures and handling of the rats were performed by trained personnel to minimize stress and discomfort.
Declarations
All procedures regarding the use and handling of animals in research were reviewed and approved by the Toho University Animal Care and User Committee (approval numbers 20-442 and 24-567) and all the experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals in research. All the performed procedures and all the reported data were in accordance with the ARRIVE guidelines.
Induction and evaluation of EAN
Rats were subcutaneously injected with 150 µg of a synthetic peptide that corresponds to amino acid residues 53–78 of bovine P2 protein (TESPFKNTEISFKLGQEFEETTADNR, Operon, Tokyo, Japan), emulsified in an equal volume of Complete Freund's adjuvant (Sigma-Aldrich, MO, USA). The injections were administered to the right footpad with rats were under light anesthesia induced by sevoflurane (Mylan, Osaka, Japan) [14].
The motor function of the rats was observed daily, and a scoring system was used to assess
paralysis of the tail, forelimbs, and left hind limb. The tail motility was scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating paralysis of the tip, 2 indicating incomplete paralysis of the entire tail, and 3 indicating complete paralysis of the entire tail. Forelimbs were scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating an inability to climb a fence using the forelimbs, 2 indicating an inability to walk, and 3 indicating complete paralysis. The left hind limb was scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating paralysis of the toe only, 2 indicating incomplete dorsiflexion of the foot joint while walking, and 3 indicating complete paralysis, where the legs were dragged when walking. The rat with shallow breathing suspected respiratory muscle paralysis were euthanized with an overdose of sevoflurane before reaching that state.
In this study, 30 rats were sacrificed across all phases, with 10 each on day 10 post-immunization (p.i.) for the subclinical to acute phase, day 14 p.i. for the early peak phase, and day 21 p.i. for the recovery phase.
Sphingosine-1-phosphate receptor 5 agonist treatment
The S1Pr5 agonist A-971432, (Cayman Chemical Co., Ltd., Michigan, USA) was in a solution of 0.5% carboxymethylcellulose (CMC) in phosphate-buffered saline (PBS) in preparation for oral administration. This solution was administered to the experimental rats, specifically those assigned to the S1Pr5 agonist group, at a daily dose of 1.0 mg/kg, starting from day 5 and continuing until day 20 p.i.. The purpose of this daily administration was to assess the effects of S1Pr5 agonist within the scope of the study.
Tissue collection
Rats were sedated using sevoflurane and underwent complete perfusion with ice-cold PBS on days 10, 14, and 21 p.i. Subsequently, the lumbar spinal cord was excised, and a portion of the cauda equina was preserved in 10% buffered formalin for histological examination. The other portion was kept in Allprotect® Tissue Reagent (Qiagen, KK, Tokyo, Japan) at -80°C in a deep freezer for subsequent analysis of mRNA and cytokines.
Sequential analysis of cytokine production related to pathogenesis of EAN
The cauda equina was subjected to enzyme-linked immunoassay (ELISA) using a 96-well plate (specifically the Rat IFN-γ and IL-10 DuoSet® ELISA Kit from biotech, USA). To achieve this, the cauda equina sample stored in Allprotect® tissue reagent was dissociated in 750 ml solution of 10 % radioimmunoprecipitation assay lysis buffer including protease inhibitor (Santa Cruz Biotechnology, CA, USA) using a bead tissue grinder (Shakeman™, Biomedical Science, Tokyo, Japan). The ELISA process was then initiated by diluting the capture antibody for each cytokine and coating each well. The plates were incubated overnight at room temperature, followed by washing to remove excess antibodies. Nonspecific sites were blocked using the reagent diluent (10% BSA dissolved in PBS), after which the plate was incubated again. Subsequently, 20 μg/ml of homogenized cauda equina solution or standards were added to the wells, covered, and incubated for two hours to allow antigen binding. After this incubation period, the plate was washed again before adding the detection antibody, followed by another incubation and washing step. Streptavidin-horseradish peroxidase was then introduced into the plate, which was incubated in the dark before being washed again. To develop color, a substrate solution was added to the plate and the reaction was stopped with a stop solution before mixing.
Finally, the absorbance was measured at 450 nm with an iMark™ Microplate Absorbance Reader (BIO-RAD, Tokyo, Japan). Cytokine concentrations are expressed in pg/mL.
Sequential analysis of messenger RNA expression of regeneration of peripheral nerve
Before RNA extraction, the cauda equina was crushed using a bead-type tissue grinder (Shakespeare Biomedical Science, Tokyo, Japan). The mRNA expression of the transcription factors C-Jun and Shh, which are related to Schwann cell regeneration, were subsequently analyzed. RNA extraction, cDNA synthesis, and mRNA expression analyses were performed in the same manner as described for Schwann cells.
Histological analysis
The cauda equina collected on day 14 p.i. was embedded in paraffin and cut into transverse sectional 5 μm thick slices. Luxol fast blue (LFB) staining and counterstaining with hematoxylin were applied to identify the areas of demyelination and infiltrating cells, respectively. Using the Hitsugi_planimetry software (https://hitsugi-edu-inst.main.jp/computer-software/Hitsugi-planimetry-manual), the ratio of demyelinated area and the number of infiltrating cells per square micrometer were quantified in 50 randomly sampling transverse sectional nerves of cauda equina from all groups.
To validate the effect of the S1Pr5 agonist on cellular infiltration, we used specific antibodies to identify T lymphocytes and macrophages with the highest precision. T lymphocytes were detected using an anti-CD3 rabbit monoclonal antibody (SP7, Nichirei Biosciences Inc., Tokyo, Japan), whereas macrophages were identified using an anti-Iba-1 rabbit polyclonal antibody (GTX100042; GeneTex Inc., Irvine, CA, USA), following the standard antigen retrieval protocol recommended by the manufacturers.
Shh was chosen as a marker for the regeneration of damaged Schwann cells because of its higher mRNA production compared to C-Jun. Anti-Shh rabbit polyclonal antibodies (St Johns Laboratory Ltd, London, UK) were applied to paraffin-embedded sections without an antigen retrieval protocol, according to the manufacturer's instructions. A peroxidase-labeled anti-rabbit IgG goat polyclonal antibody conjugated with an amino acid polymer (N-Histofine® Simple Stain MAX-PO (R)TM, Nichirei Biosciences Inc., Tokyo, Japan) was used to visualize these antibodies on tissue sections, followed by diaminobenzidine as a chromogen.
Statistical analysis
Analysis of the groups was performed with the Mann–Whitney U test, a non-parametric method. The R software (version 3.2.2; R Foundation for Statistical Computing, Vienna, Austria), utilizing the rms package [15], was employed to process the data, setting statistical significance at p < 0.05. Unless specified differently, results are expressed as mean ± SEM.