Patient information and sample collection
CSF was obtained from patients with AQP4-Ab–positive NMOSD in the acute (n = 17) or remission phase (n = 18), MS in the acute (n = 15) or remission phase (n = 6), or other neurological diseases (ONDs, n = 56). All NMOSD subjects were diagnosed according to the 2015 NMOSD diagnostic criteria, and all MS patients fulfilled the 2010 McDonald criteria. ONDs included Guillain-Barré syndrome (GBS) (n = 6), amyotrophic lateral sclerosis (ALS) (n = 27), Parkinson disease (PD) (n = 6), idiopathic normal pressure hydrocephalus (iNPH) (n = 5), cervical spondylosis (n = 5), and somatic symptom disorders (n = 7). The acute phase of NMOSD was defined as a sudden appearance of new neurological symptoms, and CSF during the acute phase was collected before or within 24 h from the start of treatment (high-dose intravenous methylprednisolone or plasmapheresis). CSF in the remission phase was collected after symptoms resolved following treatment for the acute phase. Informed consent was obtained from each patient. This study was approved by the ethics committee of Osaka University Hospital (permit number 12091-6).
Surgical procedures
Female Lewis rats (age, 8 weeks; body weight, 200–250 g) purchased from the Charles River Laboratories Japan (Yokohama, Japan) were anesthetized with a mixture of Dormicam (4 mg/kg), Vetorphale (5 mg/kg), and Domitor (4 mg/kg), and laminectomy was performed at the thoracic 10 (Th10) vertebral level, where the spinal cord was exposed. A microsyringe (75RN, Hamilton, Reno, NV, USA) was inserted 1.5 mm at Th10 and used to infuse patient-derived AQP4 recombinant antibody [12] or control IgG [Human IgG1 lambda, Nordic-MUbio BV, Susteren, Netherlands] (20 μg), 1 μL of normal human serum, and indocyanine green (Tokyo Chemical Industry, Tokyo, Japan). Total volume was 4 μL. Rats in the TNP-ATP–treated group received a mixture of 1 μg TNP-ATP (SM0740, Sigma-Aldrich, St. Louis, MO, USA). All experimental procedures were performed according to the protocols approved by the Animal Care and Use Committee of Osaka University School of Medicine and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Immunohistochemistry
HEK293 cells or HEK293 cells expressing AQP4 (HEK-AQP4) were fixed in 4% paraformaldehyde for 15 min. After incubation with 1% BSA in PBS for 30 min at room temperature, cells were incubated with anti-AQP4 IgG FITC (1:50; Santa Cruz Biotechnology; Santa Cruz, CA, USA) overnight at 4 °C. Rats were euthanized by terminal anesthesia. Spinal cords were embedded in Tissue Tek OTC compound 4583 (Sakura Finetech, Tokyo, Japan) and snap-frozen. Twenty micrometer-thick frozen tissue sections were fixed in 4% paraformaldehyde for 15 min. To stain GFAP and AQP4, after incubation with 1% BSA in PBS for 30 min at room temperature, the sections were incubated with anti-AQP4 (1:50; SAB5200112, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C. The sections were incubated with goat anti-rabbit IgG Alexa Fluor 594 (1:200; Abcam, Cambridge, UK). Then, the section was incubated with anti-GFAP Alexa Fluor 488 conjugate (1:50; 3655 Cell Signaling Technology, Beverly, MA, USA). Images were captured on a BZ-X710 microscope (Keyence, Osaka, Japan).
Behavioral studies
To assess mechanical sensitivity, calibrated von Frey filaments (Aesthesio, DanMic Global, San Jose, CA, USA) were applied to the plantar surfaces of the hindpaws of NMOSD and control rats. The rats were placed in a plastic cage with a wire mesh bottom that allowed access to their paws. Behavioral acclimatization was allowed for 1 h until cage exploration and grooming activities ceased. The area tested was the mid-plantar hindpaw. Series of von Frey monofilaments with different pressure intensities (from 1.0 to 26 g) were applied. The threshold of pain was determined as the lowest pressure filament that induced avoiding response in five out of ten applications.
Cell cultures
HEK293 cells transfected with or without M23-human AQP4 expression plasmid (GeneCopoeia, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin–streptomycin. The M23-human AQP4 plasmid was transfected into HEK293 cells by using FuGENE6 Transfection Reagent (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer’s instructions. Clones of stably transfected cells were selected by 1.0 mg/ml G418. The selected clones were confirmed to show high expression of AQP4 by western blot analysis and immunocytochemistry. Primary astrocytes were obtained from Sprague–Dawley rats as previously described [13] according to the protocols approved by the Animal Care and Use Committee of Osaka University School of Medicine and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Immunohistochemical analysis confirmed that more than 95% of collected cell population consisted of GFAP-positive astrocytes.
Quantification of ATP
HEK293 cells were seeded in 24-well culture plates and stimulated with DMEM with or without AQP4 recombinant antibody [12] containing 0, 5, or 10% normal human serum at 37 °C. Culture supernatants were collected 2 h after antibody stimulation and centrifuged at 2000×g at 4 °C for 10 min. Rat primary astrocytes were seeded in 24-well culture plates and stimulated with DMEM with or without AQP4 recombinant antibody containing 10% NHS at 37 °C. Culture supernatants were collected 2 h after antibody stimulation and centrifuged at 2000×g at 4 °C for 10 min. Supernatants were analyzed using the ATP Bioluminescence Assay Kit HS II (Roche, Basel, Switzerland). The standard samples and control groups were added to the corresponding wells. CSF samples were centrifuged at 2000×g at 4 °C for 10 min. ATP levels in CSF samples of patients were measured using the ATPlite luminescence assay system (PerkinElmer, Waltham, MA, USA). In this assay, the emitted light produced by the reaction of ATP with added luciferase and D-luciferin is proportional to the concentration of ATP. In a 96-well white plate, 50 μL of the reconstituted reagent was added to each well, which contained 100 μL of CSF supernatant centrifuged at 2000×g at 4 °C for 10 min and equilibrated at room temperature. Luminescence was measured using a Centro XS3 LB960 luminescent microplate reader (Berthold Technologies, Bad Wildbad, Germany).
Transcriptome analysis
For transcriptome analysis of spinal cord tissue, rAQP4 IgG– or control IgG–treated rats were transcardially perfused with ice-cold PBS under anesthesia with i.p. injection of a mixture of Dormicam (4 mg/kg), Vetorphale (5 mg/kg), and Domitor (4 mg/kg). Spinal cords (n = 3 each group) were cut out at the Th10 level, and total RNA was prepared using ISOGEN II (Nippongene, Toyama, Japan). RNA libraries were prepared for sequencing using the TruSeq Stranded mRNA Library Prep kit (Illumina, San Diego, CA, USA). Sequencing was performed on the Illumina HiSeq 2500 platform in a 75-bp single-end mode. Sequenced reads were mapped to the rat reference genome sequence (rn6) using TopHat ver. 2.1.1 in combination with Bowtie2 ver. 2.2.3. The raw counts were calculated using Cufflinks ver. 2.2.1. Transcriptome analysis was performed using either iDEP.91 [14], the STRING database [15], or Enrichr [16]. For principal component analysis (PCA), normalization and scaling of raw read counts were processed by the EdgeR package of iDEP.91 (filtered with 0.5 counts per million); among the six samples, 12,453 genes passed the filter. Differentially expressed genes (DEGs) were assessed by DESeq2 (false discovery rate cutoff < 0.1, fold change > 2) and visually depicted using the STRING database. Heat map of purinergic receptors was calculated with iDEP.91 by normalizing samples against the standard deviation. Using Enrichr-based WikiPathways analysis, gene set enrichment analysis was performed with upregulated DEGs of NMOSD rats that met the criterion |log2 (fold-change)| > 1. Raw data from this analysis were submitted to Gene Expression Omnibus (GEO) under accession number GSE150598.
Quantitative PCR
Total RNA of the spinal cords was obtained as described in the “Transcriptome analysis” section. Sixty nanograms RNA extracted according to the instruction protocol was reverse transcribed with SuperScript VILO (Thermo Fisher Scientific, San Jose, CA, USA) as follows: 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min. cDNA corresponding to 1ng RNA was measured with q-PCR. The q-PCR was performed using TaqMan probes according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA), and the reactions were performed using the Applied Biosystems 7900HT (Applied Biosystems, Foster City, CA, USA). The following TaqMan primers were used: Rn00580432_m1 (interleukin 1 beta; IL1B) and Rn00667869_m1 (Actin beta; Actb). The resultant data were analyzed according to the comparative cycle threshold (Ct) method (ΔΔCt), which was normalized to the expression of Actb reference genes.
Statistics
Data are shown as means ± SEM. Data assessing allodynia and HEK-AQP4 ATP assay were analyzed by linear mixed regression analysis for factorial repeated measures ANOVA model with random-effects of one within-subjects (the same rat’s paired right and left data), with post hoc Bonferroni correction. Rat astrocyte ATP assay and quantitative polymerase chain reaction analysis were analyzed by Student’s t test. CSF ATP levels of different groups were analyzed by Welch’s T test. Statistical significance was defined as P < 0.05. Asterisks in figures denote P values as follows: *P < 0.05 and **P < 0.01.